Chimeric antigen receptors against multiple HLA-G isoforms

ABSTRACT

The present invention relates to chimeric antigen receptors (CAR) against multiple but not all human leukocyte antigen (HLA-G) isoforms. More specifically, the invention concerns CARs that are specific for HLA-G β2M-free or β2M-associated immunosuppressive isoforms respectively.

This application is a continuation of International Patent ApplicationNo. PCT/EP2019/073257, filed on Aug. 30, 2019, which claims the benefitof European Patent Application No. 19305809.6, filed on Jun. 21, 2019and European Patent Application No. 18306153.0, filed on Aug. 31, 2018,the disclosure of each of which is hereby incorporated by reference inits entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:INVE_002_01US_SeqList.txt, date recorded: Oct. 22, 2020, file size:122,121 bytes).

FIELD OF THE INVENTION

The invention relates to the field of immunology, cell biology, andmolecular biology. In certain aspects, the field of the inventionconcerns immunotherapy. More particularly, the invention relates tochimeric antigen receptors (CAR) against multiple human leukocyteantigen (HLA-G) isoforms.

BACKGROUND OF THE INVENTION

For years, the foundations of cancer treatment were surgery,chemotherapy, and radiation therapy. However, in the past several years,immunotherapy has emerged as an effective tool in cancer treatment.Advances in genetic engineering have led to the design of synthetictumor targeting receptors, termed chimeric antigen receptors (CARs) thatcan be introduced into human immune cells such as T cells to redirectantigen specificity and enhance functions of effector immune cells. CARswere first developed in the mid-1980s and the interest on thesereceptors is growing since. They were first generated to bypass theintrinsic TCR specificity of expressing T cells by providing specificantigen recognition independently from HLA-peptides complexes.Prototypic single chain CARs were first described in a study by Eshharand colleagues in 1993 in which specific activation and targeting of Tcells was mediated through molecules consisting of atarget-antigen-specific antibody domain and the γ- or ζ-signalingsubunits of the Fc epsilon receptor or T-cell receptor CD3 complex,respectively (Eshhar et al., 1993, Proc Natl Acad Sci USA., 90, 720-4).Since then many groups have devised CAR molecules with singletumor-directed specificities and enhanced signaling endodomains.Nowadays, the binding domain of a CAR typically consists of anantigen-binding domain of a single-chain antibody (scFv) orantibody-binding fragment (Fab) selected from a library and comprisingthe light and heavy chain variable fragments of a monoclonal antibody(Mabs) joined by a flexible linker. The scFv retains the samespecificity and a similar affinity as the full antibody from which itwas derived and is able to specifically bind to the target of interest.CARs thus combine antigen-specificity and T cell activating propertiesin a single fusion molecule. Indeed, the scFv is linked to anintracellular signaling module that includes CD3ζ to induce T cellactivation upon antigen binding. The modular structure has been extendedfrom first-generation CARs with only a CD3ζ signaling domain to secondand third generation CARs that link the signaling endodomains such asCD28, 4-1BB, or OX40 to CD3ζ, in an attempt to mimic co-stimulation.Generally, a spacer or hinge domain serves as a linker between theendodomains and the scFV. The incorporation of such hinge domainimproves flexibility, spatial organization and/or proximity but also theexpansion of CAR cells (Qin et al, Journal of Hematology & Oncology.2017; 10:68) or tumor localization (Watanabe et al, Oncoimmunology.2016; 5(12): e1253656). It is thus critical to develop CAR comprising anoptimized hinge domain.

The CARs allow T cells to target cancers in an MHC independentmechanism. Ligand binding of a CAR differs from that of a TCR binding topeptide/MHC (pMHC) in receptor affinity, antigen density, and spatialproperties; and experimental approaches to design an optimal CAR for aspecific target molecule have relied on functional assays of transducedT cells in vitro or in human tumor xenograft models. Because it isunlikely that CARs will serially engage target molecules and cluster inorganized synapses as it is observed with TCR/pMHC recognition, it isassumed that a higher ligand density is required for CAR recognitionthan for TCRs. The optimal configuration and application of thesereceptors, besides affinity and specificity, rely on construct design,signaling domains, vector delivery systems, recipient immune-cellpopulations, and manufacturing.

While many attempts to achieve the success of CART cells for solidtumors have been undertaken results have been sometimes disappointing.The three main hurdles encountered for the application of CART celltherapies to solid tumors are (1) the identification of proper TumorAssociated Antigens (TAA), (2) the limited trafficking of adoptivelytransferred cells to tumor sites (3) the immunosuppressive effect oftumor microenvironment and (4) the potential safety issue of unwanted oruntransformed cells.

Regarding the choice of the target, the human leukocyte antigen G(HLA-G) is depicted as a molecule able to confer protection to the fetusfrom the immune system of its mother's recognition and destruction,providing fetal-maternal tolerance. Besides its physiologic functions,HLA-G was recently identified as an immune checkpoint (ICP) molecule,which inhibits the effector functions of infiltrating immune cellsubsets through the interaction with its specific receptors. HLA-G isexpressed in numerous tumor effusions of diverse origins with a highlyrestricted tissue expression. In several malignant transformations, theexpression of HLA-G by tumor cells rises dramatically, rendering themstrongly immunosuppressive. Preclinical models have shown that theexpression of HLA-G on cancer cells renders them more metastatic andsignificantly decreases patient survival (Lin A et al. Int J Cancer.2012 Jul. 1; 131(1):150-7. doi: 10.1002/ijc.26375; Lin A et al. HumImmunol. 2013 April; 74(4):439-46. doi: 10.1016/j.humimm.2012.11.021).

HLA-G is a non-classical HLA class I molecule that was first identifiedin choriocarcinoma cells. Unlike classic HLA class I molecules, HLA-G ischaracterized by a limited polymorphism and differs as well by itsexpression, structure and functions. The primary transcript of HLAG isalternatively spliced resulting in the expression of seven isoforms,where four are membrane-bound (HLA-G1, HLA-G2, HLA-G3 and HLA-G4) whilethe other three are soluble (HLA-G5, HLA-G6 and HLA-G7). HLA-G1 andHLA-G5 are the most studied isoforms and they present the typicalstructure of a classical HLA class I molecule: a heavy chain constitutedof three globular domains non-covalently bound to β2-microglobulin (β2M)and a peptide, while the other isoforms are shorter, lacking one or twodomains of the heavy chain, and should not bind β2M.

Considering that the basic criteria to develop CAR immunotherapies arethe identification of proper TAA, the accessibility of transgeniceffector cell and reversibility of the immunosuppressive tumormicroenvironment, and also taking into account the neo-expression ofHLA-G by tumor cells and its contribution to the immunosuppressiveeffect of tumor microenvironment and its role as an ICP and in immuneescape mechanisms, HLA-G is an exceptional candidate to develop newimmunotherapies able to block this molecule. Moreover, given that nostimulatory functions or cellular responses directed against allogeneicHLA-G have been reported, and that HLA-G expression istissue-restricted, the generation of cytolytic CAR cells directedagainst HLA-G would open up new possibilities in the field of cancerimmunotherapy.

WO2016/160622 discloses antibodies directed against HLA-G and their usesin CAR cells. However, as HLA-G mRNA is spliced into different proteinisoforms, there is a need to develop CARs able to specifically recognizethe different HLA-G isoforms β2M-associated HLA isoforms and smallerβ2M-free isoforms) and/or the most abundant isoforms, to eliminate mostof the cells that express the immunosuppressive isoforms, but also CARconstructs that comprise an optimized spacer domain and a selectablemarker to improve CAR cells expansion and selection.

SUMMARY OF THE INVENTION

Aiming at the largest number of HLA-G isoforms, the inventors developedseveral chimeric antigen receptors directed against HLA-G using scFvderivatives of new anti-HLA-G specific monoclonal antibodies (Mabs).These Mabs present high affinity for HLA-G and are directed to differentepitopes of the molecule, considering the heterogenicity of expressionof this molecule [Alegre et al, Eur. J. Immunol. 2013. 43: 1933-1939;Alegre E et al, 2014, Journal of Immunology Research, 2014: 657625]. Theinventors also optimized the spacer domain between the antigen bindingdomain and the endodomain to improve anti-HLA-G CAR cells efficacy. Theinventors finally designed a CAR construct comprising a cleavable linkerand a reporter that allows the selection of transduced CAR cells.

The invention provides a chimeric antigen receptor (CAR) comprising anantigen binding domain of an anti-HLA-G antibody, a transmembrane domainand an intracellular domain, wherein said CAR specifically binds one tosix HLA-G isoform(s), preferably two to five HLA-G isoforms, preferablysuch CAR allows the discrimination of HLA-G isoforms, i.e. the CAR doesnot recognize or bind all of the seven HLA-G isoforms.

In one aspect, the CAR specifically binds β2M-free HLA-G or toβ2M-associated HLA-G isoforms. Alternatively, the CAR specifically bindsHLA-G isoform selected from the group consisting of HLA-G1, HLA-G2,HLA-G5 and HLA-G6. In another aspect, the CAR specifically binds bothHLA-G1 and HLA-G5, or to both HLA-G2 and HLA-G6 and/or to bothHLA-G1/β2M-free and HLA-G5/β2M-free isoforms.

The invention particularly provides an anti HLA-G chimeric antigenreceptor (CAR) sequentially comprising from N to C terminus: (a) apeptide signal sequence, b) an anti-HLA-G antibody or an antigen bindingfragment thereof, c) optionally a spacer domain, d) a transmembranedomain, e) an intracellular domain, and optionally f) a cleavable linkerand optionally g) a truncated human CD19 domain.

In one aspect, the spacer domain comprises or consists of (i) a humanIgG4 hinge domain, (ii) a human IgG4 hinge domain and a CH3 human IgG4domain or (iii) a mutated CH2 human IgG4 domain, a human IgG4 hingedomain and a CH3 human IgG4 hinge domain. Preferably, the spacer domaincomprises or consists of the sequence set forth in (i) SEQ ID No: 25,(ii) SEQ ID No: 25 and SEQ ID No: 27, or (iii) SEQ ID No: 25, SEQ ID No:26 and SEQ ID No: 27, or a homologous sequence showing more than 80%,preferably more than 90%, still preferably more than 95% identitytherewith.

In another aspect, the signal peptide is selected from the groupconsisting of a CD8a signal peptide, a mouse Ig Kappa signal peptide, ahuman IgG4 signal peptide, an IL2 signal peptide, a human IgG2 signalpeptide and a Gaussia luc signal peptide.

In a particular aspect, the transmembrane domain is selected from CD28,CD3 and CD8 transmembrane domains, preferably the transmembrane domainis a CD28 transmembrane domain.

Preferably, the anti HLA-G CAR of the invention comprises anintracellular domain that comprises a CD3 zeta signaling domain and atleast one costimulatory domain(s) selected from CD28, 41BB, CD28, CD134,ICOS, OX40, CD149, DAP10, CD30, IL2-R, IL7r6, IL21-R, NKp30, NKp44,CD27, CD137 and DNAM-1 costimulatory domains, preferably the twocostimulatory domains are 41BB and CD28 costimulatory domains.

In one aspect, the cleavable linker is selected from the groupconsisting of P2A, T2A, E2A, B2A and F2A. In another aspect, thetruncated human CD19 domain consists of the sequence set forth in SEQ IDNo: 29. Particularly, the CAR, the anti-HLA-G antibody or antigenbinding fragment thereof, preferably a scFV, selectively binds β2M-freeHLA-G or to β2M-associated HLA-G isoforms but does not bind all HLA-Gisoforms.

In another aspect, the CAR, the anti-HLA-G antibody or antigen bindingfragment thereof, preferably a scFV, or the cell expressing CAR does notbind the alpha1 domain of HLA-G.

Preferably, the anti-HLA-G antibody or antigen binding fragment thereofis an anti-HLA-G scFv that comprises:

(a) (i) the heavy chain variable region comprises SEQ ID NO: 1 or ahomologous sequence showing more than 80%, preferably more than 90%,still preferably more than 95% identity with SEQ ID NO: 1; and (ii) thelight chain variable region comprises SEQ ID NO: 2 or a homologoussequence showing more than 80%, preferably more than 90%, stillpreferably more than 95% identity with SEQ ID NO: 2; or

(b) (i) the heavy chain variable region comprises SEQ ID NO: 3 or ahomologous sequence showing more than 80%, preferably more than 90%,still preferably more than 95% identity with SEQ ID NO: 3; and (ii) thelight chain variable region comprises SEQ ID NO: 4 or a homologoussequence showing more than 80%, preferably more than 90%, stillpreferably more than 95% identity with SEQ ID NO: 4.

Preferably, the antigen binding domain of the CAR is a scFv thatspecifically binds β2M-associated HLA-G; preferably to both HLA-G1 andHLA-G5, and the scFv comprises:

(a) (i) the heavy chain variable region comprises SEQ ID NO: 1 or ahomologous sequence showing more than 80%, preferably more than 90%,still preferably more than 95% identity with SEQ ID NO: 1; and (ii) thelight chain variable region comprises SEQ ID NO: 2 or a homologoussequence showing more than 80%, preferably more than 90%, stillpreferably more than 95% identity with SEQ ID NO: 2; or

(b) (i) the CDR (Complementarity Determining Region) 1, CDR2 and CDR3 ofthe heavy chain variable region comprises SEQ ID NO: 1; and the CDR1,CDR2 and CDR3 of the light chain variable region comprises SEQ ID NO: 2.

Alternatively, the antigen binding domain of the CAR is a scFv thatspecifically binds β2M-free HLA-G, preferably to both HLA-G2 and HLA-G6and/or to both HLA-G1/β2M-free and HLA-G5/β2M-free isoforms and the scFvcomprises:

(a) (i) the heavy chain variable region comprises SEQ ID NO: 3 or ahomologous sequence showing more than 80%, preferably more than 90%,still preferably more than 95% identity with SEQ ID NO: 3; and (ii) thelight chain variable region comprises SEQ ID NO: 4 or a homologoussequence showing more than 80%, preferably more than 90%, stillpreferably more than 95% identity with SEQ ID NO: 4; or

(b) (i) the CDR (Complementarity Determining Region) 1, CDR2 and CDR3 ofthe heavy chain variable region comprises SEQ ID NO: 3; and the CDR1,CDR2 and CDR3 of the light chain variable region comprises SEQ ID NO: 4.

Preferably, the intracellular domain of the CAR comprises a CD3 zetasignaling domain and optionally at least one costimulatory domain(s)selected from CD28, 4166, CD28, CD134, ICOS, OX40, CD149, DAP10, CD30,IL2-R, IL7r6, IL21-R, NKp30, NKp44, CD27, CD137 and DNAM-1 costimulatorydomains, preferably the two costimulatory domains are 4166 and CD28costimulatory domains.

Preferably, the transmembrane domain of the CAR is selected from CD28,CD3 and CD8 transmembrane domains, preferably the transmembrane domainis a CD28 transmembrane domain.

Preferably, the CAR further comprises a hinge region connecting theantigen binding domain to the transmembrane domain, preferably selectedin the group consisting of (i) CD28 hinge, (ii) CD8 alpha hinge, (iii) ahuman IgG4 hinge domain, (iv) a human IgG4 hinge domain and a CH3 humanIgG4 domain and (v) a mutated CH2 human IgG4 domain, a human IgG4 hingedomain and a CH3 human IgG4 hinge domain.

The invention also relates to a multispecific CAR construct whichcomprises at least two different antigen binding domains, atransmembrane domain and an intracellular domain, wherein at least oneof the antigen binding domains specifically binds HLA-G isoformsassociated with a β2M domain of HLA-G; and at least another of theantigen binding domains specifically binds HLA-G isoforms which are notassociated with a β2M domain of HLA-G.

Preferably, the multispecific CAR construct is a bispecific CARconstruct that comprises (i) a domain comprising CDR 1, 2 and 3 (HCDR1,HCDR2, HCDR3) comprising a sequence of SEQ ID NO: 11, 12 and 13,respectively, (ii) a domain comprising CDR 1, 2 and 3 (LCDR1, LCDR2,LCDR3) comprising a sequence of SEQ ID NO: 14, 15 and 16, respectively,(iii) a domain comprising CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3)comprising a sequence of SEQ ID NO: 5, 6 and 7, respectively, and (iv) adomain comprising CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) comprising asequence of SEQ ID NO: 8, 9 and 10, respectively, optionally whereineach CDR may optionally comprise 1, 2, 3 or 4 amino acid substitutions,deletions or insertions.

The invention also envisions a nucleic acid molecule encoding the CAR ofthe invention, to an expression vector, comprising the nucleic acidmolecule and to a cell comprising the CAR of the invention or thenucleic acid molecule of the invention, or the expression vector of theinvention, preferably wherein the cell is selected from a groupconsisting of a T cell, CD4⁺ T cell, CD8⁺ T cell, B cell, NK cell, NKTcell, monocyte and dendritic cell, preferably the cell being a T cell, aB cell or a NK cell.

The invention also concerns a pharmaceutical composition comprising anucleic acid molecule, an expression vector or a cell according to theinvention and optionally a pharmaceutically acceptable carrier.Preferably, the cell of the invention or the pharmaceutical compositionof the invention is for use in the treatment of cancer or for use in thetreatment of a viral infection. The cell or pharmaceutical compositionfor such uses can be administered in combination with a CAR therapy thatdoes not target HLA-G.

In one aspect, the pharmaceutical composition comprises (i) a cellcomprising a CAR specifically binding β2M-associated HLA-G; preferablyto both HLA-G1 and HLA-G5, and a cell comprising a CAR specificallybinding β2M-free HLA-G, preferably to both HLA-G2 and HLA-G6 and/or toboth HLA-G1/β2M-free and HLA-G5/β2M-free isoforms; or (ii) a cellcomprising a CAR specifically binding both HLA-G1 and HLA-G5 or toβ2M-free HLA-G isoforms, and a CAR specifically binding both HLA-G2 andHLA-G6 or to β2M-associated HLA-G isoforms.

The invention finally concerns an anti-HLA-G antibody that specificallybinds HLA-G β2M-associated isoforms, preferably both HLA-G1 and HLA-G5.Such antibody comprises:

(a) (i) the heavy chain variable region comprises SEQ ID NO: 1 or ahomologous sequence showing more than 80%, preferably more than 90%,still preferably more than 95% identity with SEQ ID NO: 1; and (ii) thelight chain variable region comprises SEQ ID NO: 2 or a homologoussequence showing more than 80%, preferably more than 90%, stillpreferably more than 95% identity with SEQ ID NO: 2; or

(b) (i) the CDR (Complementarity Determining Region) 1, CDR2 and CDR3 ofthe heavy chain variable region comprises SEQ ID NO: 1; and the CDR1,CDR2 and CDR3 of the light chain variable region comprises SEQ ID NO: 2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1C: (FIG. 1A) HLA-G receptors (FIG. 1B) Alternativesplicing of HLA-G primary transcript generates seven HLA-G isoforms, 4membrane bounds and 3 soluble isoforms (FIG. 1C) Immunosuppressivefunctions of HLA-G.

FIG. 2A-FIG. 2C: Monoclonal antibody specificity analyzed byflow-cytometry. (FIG. 2A) 15E7 monoclonal antibody is specific forβ2M-free HLA-G isoforms. (FIG. 2B) Cell surface HLA-G1 expressing celllines labeled by 15E7 antibody. (FIG. 2C) Cell surface HLA-G1 expressingcell lines labeled by LFTT-1 antibody.

FIG. 3A-FIG. 3B: (FIG. 3A) 15E7 3^(rd) CAR generation structure. (FIG.3B) LFTT-1 3^(rd) CAR generation structure.

FIG. 4A-FIG. 4B: Schematic representations of (FIG. 4A) the 3 HLA-G CARchains depending on their hinge and (FIG. 4B) the resulting HLA-G CARproteins.

FIG. 5A-FIG. 5B: (FIG. 5A) Anti-HLA-G CAR 15E7 and LFTT-1 expressions onJurkat and the murine NKT 1.2 cell lines were analyzed byflow-cytometry. (FIG. 5B) HLA-G CAR 15E7 and LFTT-1 cell surfaceexpression was determined by immunofluorescence.

FIG. 6A-FIG. 6D: (FIG. 6A) Principle of activation assays. (FIG. 6B)HLA-G1 binding by 15E7 (left panel) and LFTT-1 (right panel) antibodies.(FIG. 6C) Representative figure of effector HLA-G CAR cell activation.(FIG. 6D) Effector HLA-G CAR cells were strongly activated in presenceof HLA-G expressing target cells.

FIG. 7A-FIG. 7B: CAR specificity analyzed by flow-cytometry. (FIG. 7A)Jurkat HLA-G CAR 15E7 were strongly activated in presence ofHLA-G/β2M^(neg) expressing cells. (FIG. 7B) NKT 1.2 HLA-G CAR LFTT-1were strongly activated in presence of Jeg-3 cell line.

FIG. 8A-FIG. 8B: Cytolytic function of HLA-G CART cells was investigatedon K562, K562-HLA-G1 and JEG-3 tumor cells through (FIG. 8A) tumor celllysis was investigated and (FIG. 8B) CD107a expression on effector CARscells.

FIG. 9: IFN-γ secretion of HLA-G CARs cells was investigated afterco-incubation with K562, K562-HLA-G1 and JEG-3 tumor cells.

FIG. 10A-FIG. 10B: In vivo anti-tumor functions of HLA-G CAR-T cells(FIG. 10A) Scheme of in vivo experiment in mice with HLA-G CAR-T cells(FIG. 10B) CAR-T cells cytotoxicity against HLA-G tumor cells in vivo inNGS mice.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention relates to a chimeric antigen receptor (CAR)comprising an extracellular domain, mostly constituted by the antigenbinding domain of an anti-HLA-G specific antibody, optionally a hingedomain comprising or consisting of (i) a human IgG4 hinge domain, (ii) ahuman IgG4 hinge domain and a CH3 human IgG4 domain or (iii) a mutatedCH2 human IgG4 domain, a human IgG4 hinge domain and a CH3 human IgG4hinge domain, a transmembrane domain, an intracellular domain thatcomprises one, two or three co-stimulatory structures, depending on thegeneration of the CAR design, optionally a cleavable linker andoptionally a reporter.

Particularly, the CAR according to the invention specifically binds toone to six, preferably two to five HLA-G isoform(s), more preferablyselected from HLA-G1, HLA-G2, HLA-G5 and HLA-G6.

The invention also relates to a CAR that specifically binds to bothHLA-G1 and HLA-G5 or to both HLA-G2 and HLA-G6 isoforms.

It is further provided a multispecific CAR construct, preferably abispecific CAR construct that comprises one domain that recognizes HLA-Gisoforms that are free of β2M, preferably both HLA-G2 and HLA-G6 and/orboth HLA-G1/β2M free and HLA-G5/β2M free isoforms; and one domain thatrecognizes HLA-G isoforms associated β2M, preferably HLA-G1 and HLA-G5isoforms.

In one aspect, the invention further relates to a CAR that specificallybinds to HLA-G isoforms that are free of β2M (beta-2-microglobulin). Inanother aspect, the invention further relates to a CAR that specificallybinds to HLA-G isoforms associated to β2M.

It particularly relates to a monoclonal antibody or a single chainvariable fragment (scFv) molecule that specifically binds to both HLA-G1and HLA-G5 or both HLA-G2 and HLA-G6 isoforms. It also relates to amonoclonal antibody or a single chain variable fragment (scFv) moleculethat specifically binds to HLA-G isoforms that are free of β2M,preferably to both HLA-G2 and HLA-G6 and/or to both HLA-G1/β2M free andHLA-G5/β2M free isoforms. It further relates to a monoclonal antibody ora single chain variable fragment (scFv) molecule that specifically bindsto HLA-G isoforms associated β2M, preferably HLA-G1 and HLA-G5 isoforms.

The invention also concerns nucleic acid constructs or vectorscontaining the nucleic acid construct that can be transduced into acell, preferably an immune cell such as a T cell, thereby creating arecombinant immune cell engineered to express the encoded CAR. Alsoprovided are cells that are transduced to express the CAR of theinvention, cell populations, and pharmaceutical compositions containingthe cells expressing CAR.

The invention may particularly relate to an immune cell that expressestwo different CAR, particularly a CAR that specifically binds to HLA-Gisoforms that are free of β2M, preferably to both HLA-G2 and HLA-G6and/or both HLA-G1/β2M free and HLA-G5/β2M free isoforms; and a CAR thatspecifically binds to HLA-G isoforms associated β2M, preferably HLA-G1and HLA-G5 isoforms.

Among the compositions are pharmaceutical compositions and formulationsfor administration, such as for adoptive cell therapy. Also provided aremethods for preparing CAR expressing cells and administering the cellsand compositions to subjects, e.g., patients.

Abbreviations

APC: Antigen Presenting Cell MFI: Mean Fluorescence Intensity β2M:β-2-Microglobulin MOI: Multiplicity Of Infection CAR: Chimeric AntigenReceptor NK: Natural Killer CTL: Cytotoxic T Lymphocyte MHC: MajorHistocompatibility Complex DC: Dendritic Cell PBS: Phosphate BufferedSaline HLA-G: Human Leukocyte Antigen G PBMC: Peripheral BloodMononuclear Cells ICP: Immune Checkpoint scFv: single-chain variableFragment ITAM: Immunoreceptor Tyrosine-based SD: Standard DeviationActivation Motif TAA: Tumor Associated Antigen Mab(s): MonoclonalAntibody(ies) WT: wild-type

Definitions

To facilitate the understanding of the invention, a number of terms aredefined below. The terms “Chimeric antigen receptor” (CAR), “engineeredcell receptor”, “chimeric cell receptor”, or “chimeric immune receptor”(ICR) as used herein refer to engineered receptors, which graft anantigen binding specificity onto immune cells (e.g. T cells or NKcells), thus combining the antigen binding properties of the antigenbinding domain with the immunogenic activity of the immune cell, such asthe lytic capacity and self-renewal of T cells. Particularly, a CARrefers to a fused protein comprising an extracellular domain able tobind an antigen, a transmembrane domain, optionally a hinge domain andat least one intracellular domain. The terms “extracellular domain ableto bind an antigen”, “external domain”, “ectodomain” and “antigenbinding domain” are used interchangeably herein and mean anyoligopeptide or polypeptide that can bind to a targeted antigen (e.g.HLA-G isoform(s)). Particularly, the term “antigen binding domain” or“antigen-specific targeting domain” as used herein refers to the regionof the CAR which targets and binds to specific antigens, for exampleHLA-G antigen. When a CAR is expressed in a host cell, this domain formsthe extracellular domain (ectodomain) of the receptor. The antigenbinding domain of a CAR typically derives from an antibody and mayconsist of an antigen-binding domain of a single-chain antibody (scFv)or antigen-binding fragments (Fab). The terms “intracellular domain”,“internal domain”, “cytoplasmic domain” and “intracellular signalingdomain” are used interchangeably herein and mean any oligopeptide orpolypeptide known to function as a domain that transmits a signal thatcauses activation or inhibition of a biological process in a cell. Theintracellular signaling domain may generate a signal that promotes animmune effector function of the cell transduced with a nucleic acidsequence comprising a CAR, e.g. cytolytic activity and helper activity,including the secretion of cytokines. The term “transmembrane domain”means any oligopeptide or polypeptide known to span the cell membraneand that can function to link the extracellular and signaling domains.This may be a single alpha helix, a transmembrane beta barrel, abeta-helix of gramicidin A, or any other structure. Typically, thetransmembrane domain denotes a single transmembrane alpha helix of atransmembrane protein, also known as an integral protein.

A chimeric antigen receptor may optionally comprise a “hinge domain”which serves as a linker between the extracellular and transmembranedomains. As used herein the terms “hinge”, “spacer”, or “linker” refersto an amino acid sequence of variable length typically encoded betweentwo or more domains of a polypeptide construct to confer for exampleflexibility, improved spatial organization and/or proximity.

As used herein, the term “cleavable linker” refers to a peptide chain ofvariable length that can be proteolytically cleaved or digested byproteases or enzymes or that self-cleaves. After cleavage of the peptidelinker, its integrity is generally compromised and results to theseparation of the domains located on either side of the cleavable linker(C-terminus and N-terminus). A “cleavable linker” generally comprises acleavage site. As used herein, the term “cleavage site” refers to aspecific sequence of amino acids that can be cleaved specifically by acleavage agent, such as a protease, or that self-cleaves. The term“linker” as used in the context of a scFv refers to a peptide linkerthat consists of amino acids such as glycine and/or serine residues usedalone or in combination, to link variable heavy and variable light chainregions together.

A chimeric antigen receptor may optionally comprise a signal peptide.The terms “signal peptide” “targeting signal”, “localization signal”,“transit peptide” or “leader sequence” refer to a short peptide presentat the N-terminus of the majority of newly synthesized proteins that aredestined towards the secretory pathway. The core of the signal peptidemay contain a long stretch of hydrophobic amino acids. The signalpeptide may or may not be cleaved from the mature polypeptide.

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, polyclonal antibodies, multispecific antibodies,human antibodies, humanized antibodies, camel antibodies, chimericantibodies, antigen binding fragments such as single-chain variablefragment (scFv), single chain antibodies, single domain antibodies,antigen-binding fragments (Fab), F(ab′) fragments, disulfide-linkedvariable fragment (sdFv), intrabodies, nanobodies, and epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG₂, IgG₃, IgG₄, IgA1and IgA₂) or subclass. Unless specifically noted otherwise, the term“antibody” includes intact immunoglobulins and “antibody fragments” or“antigen binding fragments” that specifically bind to a molecule ofinterest (or a group of similar molecules of interest such as HLA-Gisoforms) to the substantial exclusion of binding to other molecules.The term “antibody” also includes genetically engineered forms such aschimeric antibodies (for example, humanized murine antibodies),heteroconjugate antibodies (such as bispecific antibodies). Preferably,the term antibody refers to a monoclonal antibody, even more preferablyto a scFv derived from a monoclonal antibody.

In terms of structure, an antibody may have heavy (H) chains and light(L) chains interconnected by disulfide bonds. There are two types oflight chain, lambda (λ) and kappa (κ). Each heavy and light chaincontains a constant region and a variable region (or “domain”). Lightand heavy chain variable regions contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The framework regions act to form a scaffold that provides,for positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The CDRs are primarily responsible forbinding to an epitope of an antigen. The CDRs of each chain aretypically referred to as CDR1, CDR2, and CDR3, numbered sequentiallystarting from the N-terminus. The VL and VH domain of the antibodyaccording to the invention may comprise four framework regions or“FR's”, which are referred to in the art and herein as “Framework region1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3”or “FR3”; and as “Framework region 4” or “FR4”, respectively. Theseframework regions are interrupted by three complementary determiningregions or “CDR's”, which are referred to in the art as “ComplementarityDetermining Region 1” or “CDR1”; as “Complementarity Determining Region2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3”,respectively. These framework regions and complementary determiningregions are preferably operably linked in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxyterminus).

An “antibody heavy chain” as used herein, refers to the larger of thetwo types of polypeptide chains present in antibody conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in antibody conformations, κ andλ light chains refer to the two major antibody light chain isotypes.

The term “scFv” refers to a protein comprising at least one antibodyfragment comprising a variable region of a light chain and at least oneantibody fragment comprising a variable region of a heavy chain, whereinthe light and heavy chain variable regions are contiguously linked,e.g., via a synthetic linker, e.g., a short flexible polypeptide linker,and capable of being expressed as a single chain polypeptide, andwherein the scFv retains the specificity of the intact antibody fromwhich it is derived. Unless specified, as used herein a scFv may havethe VL and VH variable regions in either order, e.g., with respect tothe N-terminal and C-terminal ends of the polypeptide, the scFv maycomprise VL-linker-VH or may comprise VH-linker-VL. The linker maycomprise portions of the framework sequences.

The terms “derive from” and “derived from” as used herein refers to acompound having a structure derived from the structure of a parentcompound or protein and whose structure is sufficiently similar to thosedisclosed herein and based upon that similarity, would be expected byone skilled in the art to exhibit the same or similar properties,activities and utilities as the claimed compounds. For example, a scFvderived from a monoclonal antibody refers to an antibody fragment thatshares the same properties that the monoclonal antibody, e.g. sharesidentical or similar VH and VL and/or recognizes the same epitope.

As used herein, the term “antigen” refers to a compound, composition, orsubstance that may be specifically bound by the products of specifichumoral or cellular immunity, such as an antibody molecule, a T-cellreceptor or a CAR. It is readily apparent that the present inventionincludes intact antigen and antigen fragment thereof. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid. Preferably the term “antigen” refers herein to HLA-G,particularly to isoforms of HLA-G.

As used herein, the term “HLA-G” and “Human leukocyte antigen G” refersto a specific molecule associated with this name and any other moleculesthat have analogous biological function that share at least 80% aminoacid sequence identity, preferably 90% sequence identity, morepreferably at least 95% sequence identity with HLA-G, including but notlimited to any one of its several isoforms, including by not limited tomembrane-bound isoforms (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4), solubleisoforms (e.g., HLA-G5, HLA-G6, HLA-G7), and soluble forms generated byproteolytic cleavage of membrane-bound isoforms (e.g. sHLA-G1). Examplesof the HLA-G sequences are provided here below.

As used herein, “bind” or “binding” refer to peptides, polypeptides,proteins, fusion proteins and antibodies (including antibody fragments)that recognize and contact an antigen. Preferably, it refers to anantigen-antibody type interaction. By “specifically bind” or“immunospecifically bind” it is meant that the antibody recognizes aspecific antigen, but does not substantially recognize or bind othermolecules in a sample. In some instances, the terms “specific binding”or “specifically binding,” can be used in reference to the interactionof an antibody, a protein, or a peptide with a second chemical species,to mean that the interaction is dependent upon the presence of aparticular structure (e.g., an antigenic determinant or epitope). Asused herein, the term “specific binding” means the contact between anantibody and an antigen with a binding affinity of at least 10⁻⁶ M. Incertain aspects, antibodies bind with affinities of at least about 10⁻⁷M, and preferably 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M.

The term “antibody that specifically binds to an HLA-G isoform” or “CARthat specifically binds to an HLA-G isoform” and analogous terms, asused herein, refer to antibodies or antibody fragments that specificallyrecognize one or several HLA-G isoform(s) and do not or weakly recognizeother antigens (including other HLA-G isoforms). Preferably, antibodiesor antibody fragments that specifically bind to one or several HLA-Gisoform(s) have a higher affinity to this or these HLA-G isoform(s) or afragment thereof when compared to the affinity to other antigens orfragments thereof, including other HLA-G isoforms.

The affinity of an antibody can be a measure of its binding with aspecific antigen at a single antigen-antibody site and is in essence thesummation of all the attractive and repulsive forces present in theinteraction between the antigen-binding site of an antibody and aparticular epitope. The affinity of an antibody to a particular antigen(e.g. HLA-G isoform(s)) may be expressed by the equilibrium constant Kof dissociation, defined by the equation Kd=[Ag][Ab]/[Ag Ab], whichrepresents the affinity of the antibody-combining site; where [Ag] isthe concentration of free antigen (M), [Ab] is the concentration of freeantibody (M) and [Ag Ab] is the concentration (M) of theantigen-antibody complex. Where the antigen and antibody react stronglytogether there will be very little free antigen or free antibody, andhence the equilibrium constant or affinity of the antibody will be low.The average affinity for antibodies is a of at least 10⁻⁶ M. In certainaspects, the antigen binding domain of the CAR bind one to six,preferably two to five, HLA-G isoform(s) with affinities of at leastabout 10⁻⁶ M, and preferably at least 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M.The binding affinity can be measured by any method available to theperson skilled in the art, in particular by surface plasmon resonance(SPR).

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell,and the like) can specifically bind with a cognate binding partner(referred to herein as a “stimulatory molecule”) on a T cell, therebymediating a primary response by the T cell, including, but not limitedto, activation, initiation of an immune response, proliferation, and thelike.

The term “co-stimulatory ligand” as used herein, includes a molecule onan antigen presenting cell (e.g., an APC, dendritic cell, B cell, andthe like) that specifically binds with a cognate binding partner(referred to herein as a “stimulatory molecule”) on a T cell, therebyproviding a signal which, in addition to the primary signal provided by,for instance, binding of a TCR/CD3 complex with an MHC molecule loadedwith peptide, mediates a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like.

As used herein, a “co-stimulatory molecule” refers to a moleculeexpressed by an immune cell (e.g., T cell, NK cell, B cell) thatprovides the cytoplasmic signaling sequence(s) that regulates activationof the immune cell in a stimulatory way for at least some aspect of theimmune cell signaling pathway. In one aspect, the signal is a primarysignal that is initiated by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, and which leads to mediationof a T cell response, including, but not limited to, proliferation,activation, differentiation, and the like. A primary cytoplasmicsignaling sequence (also referred to as a “primary signaling domain”)that acts in a stimulatory manner may contain a signaling motif which isknown as an immunoreceptor tyrosine-based activation motif or ITAM.Particularly this term refers to the cognate binding partner on a T cellthat specifically binds with a co-stimulatory ligand present on anantigen presenting cell, thereby mediating a co-stimulatory response bythe T cell, such as, but not limited to, proliferation activation,differentiation, and the like.

A “stimulatory molecule,” as used herein, means a molecule on a T cellthat specifically binds with a cognate stimulatory ligand present on anantigen presenting cell.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation activation, differentiation, and the like, and/orupregulation or downregulation of key molecules.

By the term “stimulation” or “stimulatory” is meant a primary responseinduced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex)with its cognate ligand thereby mediating a signal transduction event,such as, but not limited to, signal transduction via the TCR/CD3complex. Stimulation can mediate altered expression of certainmolecules, such as downregulation of TGF-β, and/or reorganization ofcytoskeletal structures, and the like.

A “reporter” or “selectable marker” as used herein, refers to apolynucleotide or polypeptide that allows the detection and/or theselection of expressing cells from the population of cells sought to betransfected, particularly a cell transfected with a CAR construct. Whenlinked to a particular construct, it allows to establish the presenceand/or the quantification of such construct in a cell.

“Immune cells” as used herein refers to cells involved in innate andadaptive immunity for example such as white blood cells (leukocytes)which are derived from hematopoietic stem cells (HSC) produced in thebone marrow, lymphocytes (T cells, B cells, natural killer (NK) cellsand natural killer T cells (NKT)) and myeloid-derived cells (neutrophil,eosinophil, basophil, monocyte, macrophage, dendritic cells). Inparticular, the immune cell can be selected in the non-exhaustive listcomprising B cells, T cells, in particular CD4⁺ T cells and CD8⁺ Tcells, NK cells, NKT cells, APC cells, dendritic cells and monocytes.

According to the present invention, the donor and the recipient of theimmune cells, preferably T cells, can be a single individual ordifferent individuals, for example, autologous, allogeneic or xenogeneicindividuals. As used herein, the term “autologous” refers to cells ortissues obtained from an individual and later transplanted back into thesame individual. As used herein, the term “allogeneic” refers to cellsor tissues obtained from different individuals of the same species,where the donor and recipient are not genetically identical. With regardto the present disclosure, an allogeneic cell transplant or tissue graftinvolves transplantation of cells or tissues where the donor andrecipient are different individuals of the same species. The term“xenogeneic” means that which is derived or obtained from an organism ofa different species. With regard to the present disclosure, a xenogeneiccell transplant or tissue graft involves transplantation of cells ortissues where the donor and recipient are different individuals ofdifferent species.

The term “treatment” refers to any act intended to ameliorate the healthstatus of patients such as therapy, prevention, prophylaxis andretardation of the disease or of the symptoms of the disease. Itdesignates both a curative treatment and/or a prophylactic treatment ofa disease. A curative treatment is defined as a treatment resulting incure or a treatment alleviating, improving and/or eliminating, reducingand/or stabilizing a disease or the symptoms of a disease or thesuffering that it causes directly or indirectly. A prophylactictreatment comprises both a treatment resulting in the prevention of adisease and a treatment reducing and/or delaying the progression and/orthe incidence of a disease or the risk of its occurrence. In certainembodiments, such a term refers to the improvement or eradication of adisease, a disorder, an infection or symptoms associated with it. Inother embodiments, this term refers to minimizing the spread or theworsening of cancers. Treatments according to the present invention donot necessarily imply 100% or complete treatment. Rather, there arevarying degrees of treatment of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect.

As used herein, the terms “disorder” or “disease” refer to theincorrectly functioning organ, part, structure, or system of the bodyresulting from the effect of genetic or developmental errors, infection,poisons, nutritional deficiency or imbalance, toxicity, or unfavourableenvironmental factors. Preferably, these terms refer to a healthdisorder or disease e.g. an illness that disrupts normal physical ormental functions. More preferably, the term disorder refers to immuneand/or inflammatory diseases that affect animals and/or humans, such ascancer.

The term “immune disease”, as used herein, refers to a condition in asubject characterized by cellular, tissue and/or organ injury caused byan immunologic reaction of the subject to its own cells, tissues and/ororgans. The term “inflammatory disease” refers to a condition in asubject characterized by inflammation, e.g., chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body.

The term “adoptive cell therapy” or “adoptive T cell therapy” or “ACT”as used herein means the transfer of cells into a patient, where thecells have been engineered to or otherwise altered prior to transferinto the subject. An example of ACT is the harvesting from a subject'sblood or tumor, an immune cell, such as a T cell. These immune cells arethen stimulated ex vivo, in culture and expanded. The cells are thentransduced with one or more nucleic acid constructs that allow the cellto express new molecules, such as a CAR, providing the engineered immunecells with a new mechanism for combating a disease, for instance acancer. In some instances, the CAR comprises an antigen binding domainthat specifically recognizes an antigen expressed by a tumor or cancer,such as HLA-G. Typical immune cells utilized in ACT procedures includetumor-infiltrating lymphocytes (TILs) or T cells. Immune cells used inACT can be derived from the patient/subject themselves, or from auniversal donor. ACT may also be accompanied by the optional step oflymphodepletion of the subject's own lymphocytes that may compete withthe recombinant cells infused back into the subject.

The terms “CAR-therapy” or “CAR cell therapy” are used interchangeablyherein and refer to a type of treatment in which immune cells aremodified to express a CAR to prevent or treat a disease, for examplesuch as viral infection or cancer. Such immune cell can be autologous orallogeneic. The immune cells can for example be B lymphocyte, Tlymphocyte, natural killer cell or natural killer T cell and the like.For example, CAR cell therapy is provided by the acquisition of immunecells from a patient, transfecting the immune cells with CAR genes thatallows the expression of a CAR, such CAR being directed against anantigen involved in the patient's disease, expanding the modified immunecell population, and reinfusing the cells back into the patient. Theterm “anti-tumor effect” as used herein, refers to a biological effectwhich can be manifested by a decrease in tumor volume, a decrease in thenumber of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of cells and CARs of the inventionin prevention of the occurrence of tumor in the first place or to limitmetastasis formation.

The terms “cytolytic” or “cytotoxic” as used herein refer to the resultof the immune response mediated by cytotoxic cell such as T cell or NKcells and NKT cells that leads to the death (e.g. by apoptosis) of atargeted cell.

As used herein, the term “subject”, “host”, “individual,” or “patient”refers to human and veterinary subjects particularly to an animal,preferably to a mammal, even more preferably to a human, including adultand child. However, the term “subject” also encompasses non-humananimals, in particular mammals such as dogs, cats, horses, cows, pigs,sheep and non-human primates, among others.

As used herein, the phrase “side effects” encompasses unwanted andadverse effects of a prophylactic or therapeutic agent. Side effects arealways unwanted, but unwanted effects are not necessarily adverse. Anadverse effect from a therapy (e.g., a prophylactic or therapeuticagent) might be harmful, uncomfortable, or risky. Undesired effectstypically experienced by patients are numerous and known in the art.

The term “in combination” as used herein refers to the use of more thanone therapy (e.g., prophylactic and/or therapeutic agents). The use ofthe term “in combination” does not restrict the order in which therapies(e.g., prophylactic and/or therapeutic agents) are administered to asubject with a disease or disorder.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

As used herein, a “pharmaceutical or veterinary composition” refers to apreparation of one or more of the active agents, such as comprising anantigen binding domain of an anti-HLA-G antibody according to theinvention, with optional other chemical components such asphysiologically suitable carriers and excipients. The purpose of apharmaceutical or veterinary composition is to facilitate administrationof the active agent to an organism. Compositions of the presentinvention can be in a form suitable for any conventional route ofadministration or use. In one embodiment, a “composition” typicallyintends a combination of the active agent, e.g., compound orcomposition, and a naturally-occurring or non-naturally-occurringcarrier, inert (for example, a detectable agent or label) or active,such as an adjuvant, diluent, binder, stabilizer, buffers, salts,lipophilic solvents, preservative, adjuvant or the like and includepharmaceutically acceptable carriers.

An “acceptable vehicle” or “acceptable carrier” as referred to herein,is any known compound or combination of compounds that are known tothose skilled in the art to be useful in formulating pharmaceutical orveterinary compositions.

A “therapeutically effective amount” is an amount which, whenadministered to a subject, is the amount of active agent that is neededto treat the targeted disease or disorder, or to produce the desiredeffect. The “effective amount” will vary depending on the agent(s), thedisease and its severity and the age, weight, and characteristics of thesubject to be treated.

As used herein, the term “medicament” refers to any substance orcomposition with curative or preventive properties against disorders ordiseases.

The term “transfected” or “transformed” or “transduced” are usedinterchangeably herein and are applied to the production of chimericantigen receptor cells and particularly refer to the process whereby aforeign or exogenous nucleotide sequence is introduced into a cell. Theexogenous nucleic acid may be introduced stably or transiently into thehost cell. A “transfected” or “transformed” or “transduced” cell is onewhich has been transfected, transformed or transduced with exogenousnucleic acid. In some embodiments, this transduction is performed via avector, preferably a lentiviral vector.

As used herein, the terms “nucleic acid construct” and “vector” areequivalent and refer to a nucleic acid molecule that serves to transfera passenger nucleic acid sequence, such as DNA or RNA, into a host cell.A vector may comprise an origin of replication, a selectable marker, andoptionally a suitable site for the insertion of a sequence or gene. Avector can be either a self-replicating extrachromosomal vector or avector which integrates into a host genome. It can also compriseexpression elements including, for example, a promoter, the correcttranslation initiation sequence such as a ribosomal binding site and astart codon, a termination codon, and a transcription terminationsequence. A nucleic acid construct may also comprise other regulatoryregions such as enhancers, silencers and boundary elements/insulators todirect the level of transcription of a given gene. Vectors capable ofdirecting the expression of genes and/or nucleic acid sequence to whichthey are operatively linked can also be referred to herein as“expression vectors”. There are several common types of vectorsincluding nucleic acid constructs, phagemids, virus genomes, cosmids andartificial chromosomes. The nucleic acid construct can be a vector forstable or transient expression of a gene or sequence. The nucleic acidconstruct may comprise a nucleic acid construct origin of replication(ori). Particularly, the nucleic acid construct may be designed forgenetic transfer between different hosts, including but not limited to aplasmid, a virus, a cosmid, a phage, a BAC, a YAC. Such vectors may beprepared from commercially available vectors or produced for examplefrom baculoviruses, retroviruses, adenoviruses or AAVs according totechniques known in the art. Preferably, these terms refer to alentiviral vector.

As used herein, the term “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. The expression level of a gene may bedetermined by measuring the amount of mRNA or protein in a cell ortissue sample. In one aspect, the expression level of a gene from onesample may be directly compared to the expression level of that genefrom a control or reference sample. In another aspect, the expressionlevel of a gene from one sample may be directly compared to theexpression level of that gene from the same sample followingadministration of a compound.

The term “functional variant” or “biological equivalent” are usedinterchangeably as used herein and refer to a polypeptide (CAR orprotein) having substantial or significant sequence identity orsimilarity to a parent polypeptide, which functional variant retains thebiological activity of the polypeptide of which it is a variant. Unlessspecifically recited herein, it is contemplated that any polynucleotide,polypeptide or protein mentioned herein also includes equivalentsthereof. For example, an equivalent intends at least about 70% homologyor identity, or at least 80% homology or identity and alternatively, orat least about 85%, or alternatively at least about 90%, oralternatively at least about 95%, or alternatively 98% percent homologyor identity and exhibits substantially equivalent biological activity tothe reference protein, polypeptide or nucleic acid. Alternatively, whenreferring to polynucleotides, an equivalent thereof is a polynucleotidethat hybridizes under stringent conditions to the referencepolynucleotide or its complement. When related to antibodies, the term“equivalent” or “biological equivalent” means that the ability of theantibody to specifically bind its epitope protein or fragment thereof asmeasured by ELISA or other suitable methods is similar or conserved.Biologically equivalent antibodies include, but are not limited to,those antibodies, peptides, antibody fragments, antibody variant,antibody derivative and antibody mimetics that bind to the same epitopeas the reference antibody. When related to CARs, functional variantsencompass, for example, those variants of the CARs described herein (theparent CAR) that retain the ability to recognize target cells to asimilar extent, the same extent, or to a higher extent, as the parentCAR. In reference to the parent CAR, the functional variant can, forinstance, be at least about 50%, 75%, 80%, 90%, 98% or more identical inamino acid sequence to the parent CAR.

By “variant” or “equivalent”, it is also meant a polypeptide sequencethat differs from that of a parent polypeptide sequence by virtue of atleast one amino acid modification. For instance, in the context of theinvention, a variant may be a variant of a monoclonal antibody orfragment thereof or a variant of a CAR. Typically, a variant comprisesfrom 1 to 50 amino acid modifications, preferably from 1 to 40 aminoacid modifications. In particular, the variant may have from 1 to 30amino acid changes, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aminoacid changes as compared to its parent. The variants may comprise one orseveral amino acid substitutions, and/or, one or several amino acidinsertions, and/or one or several amino acid deletions. In someembodiments, the variant may comprise one or several conservativesubstitutions, e.g. as shown hereabove. In some further embodiments, thevariant of an antibody or antigen binding fragment thereof, particularlya scFv, may comprise one or several amino acid modifications in the CDRdomains of the parent Mab. In some other embodiments, the variant of theparent Mab may comprise one or several amino acid modifications in atleast one framework domain.

By “parent polypeptide” or “polypeptide parent”, as used herein, it ismeant an unmodified polypeptide that is subsequently modified togenerate a variant. In the context of the invention, the parentpolypeptide may be an antibody, preferably a monoclonal antibody, evenmore preferably a scFv. As used herein, “homology”, “identity” or“similarity”, when used in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences or subsequencesthat are the same or have a specified percentage of nucleotides or aminoacid residues that are the same, e.g., at least 60% identity, preferablyat least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or higher identity over a specified region (e.g.,nucleotide sequence encoding an antibody described herein or amino acidsequence of an antibody described herein). Homology can be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences. Theterm “percentage of identity” in relation to sequences designates thelevel of identity or homology between said sequences and may bedetermined by techniques known per se in the art. Typically, thepercentage of identity between two nucleic acid sequences is determinedby means of computer programs such as GAP provided in the GCG programpackage (Program Manual for the Wisconsin Package, Version 8, August1996, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48, 443-453). With settings adjusted to e.g., DNA sequences(particularly: GAP creation penalty of 5.0 and GAP extension penalty of0.3), nucleic acid molecules may be aligned to each other using thePileup alignment software available as part of the GCG program package.The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. One, non-limiting exampleof a mathematical algorithm utilized for the comparison of two sequencesare those described in Current Protocols in Molecular Biology (Ausubelet al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. or thealgorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A.87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl.Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated intothe NBLAST and XBLAST programs of Altschul et al, 1990, J. Mol. Biol.215:403. BLAST nucleotide searches can be performed with the NBLASTnucleotide program parameters set, e.g., for score=100, wordlength=12 toobtain nucleotide sequences homologous to a nucleic acid molecule of thepresent invention. BLAST protein searches can be performed with theXBLAST program parameters set, e.g., to score-50, wordlength=3 to obtainamino acid sequences homologous to a protein molecule of the presentinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al, 1997, NucleicAcids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to performan iterated search which detects distant relationships between molecules(Id). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferrednon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM 120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example. Preferably, default parameters are usedfor alignment. A preferred alignment program is BLAST, using defaultparameters. In particular, preferred programs are BLASTN and BLASTP,using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.For comparing two amino acid sequences, one can use, for example, thetool “Emboss needle” for pairwise sequence alignment of proteinsproviding by EMBL-EBI and available on:www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=emboss_needle&context=protein,using default settings: (I) Matrix: BLOSUM62, (ii) Gap open: 10, (iii)gap extend: 0.5, (iv) output format: pair, (v) end gap penalty: false,(vi) end gap open: 10, (vii) end gap extend: 0.5.

Sequence identity between nucleotide or amino acid sequences can bedetermined by comparing an alignment of the sequences. When anequivalent position in the compared sequences is occupied by the samebase or amino acid, then the molecules are identical at that position.Scoring an alignment as a percentage of identity is a function of thenumber of identical amino acids or bases at positions shared by thecompared sequences. When comparing sequences, optimal alignments mayrequire gaps to be introduced into one or more of the sequences to takeinto consideration possible insertions and deletions in the sequences.Sequence comparison methods may employ gap penalties so that, for thesame number of identical molecules in sequences being compared, asequence alignment with as few gaps as possible, reflecting higherrelatedness between the two compared sequences, will achieve a higherscore than one with many gaps. Calculation of maximum percent identityinvolves the production of an optimal alignment, taking intoconsideration gap penalties.

The terms also include sequences that have deletions and/or additions,as well as those that have substitutions, particularly conservativesubstitution. Preferably, identity exists over a region that is at leastabout 25 amino acids or nucleotides in length, or more preferably over aregion that is at least 50-100 amino acids or nucleotides in length. An“unrelated” or “non-homologous” sequence shares less than 50% identity,or alternatively less than 40% identity, preferably less than 30% withone of the sequences disclosed herein.

HLA-G Antigen

“HLA-G” designates the Human leukocyte antigen G which includes at leastseven isoforms. Its expression is mainly restricted to the feto-maternalinterface on the extravillous cytotrophoblast; to placenta, amnion; to afew healthy adult tissues such as thymus, cornea, bronchial epithelialcells, and pancreas; and to different types of cells such as mesenchymalstem cells, a few activated monocytes, and erythroid and endothelialprecursors. The soluble HLA-G is also found in body fluids such asplasma, cerebrospinal fluid, malignant ascites, pleural effusions, andsperm. Although the HLA-G gene is not active in some tissues, itsexpression can be induced by certain molecules such as progesterone oranticancer drugs. Furthermore, this molecule can also be neo-expressedas well in pathological conditions such as cancer, multiple sclerosis,inflammatory diseases, and viral infections or after allograft. SolubleHLA-G (sHLA-G) can be detected in the serum/plasma of individuals.

HLA-G differs from classical HLA class I molecules by its low geneticdiversity, a tissue-restricted expression, the existence of sevenisoforms, and immuno-inhibitory functions. This molecule exerts animmuno-inhibitory function through direct binding to three inhibitoryreceptors: leukocyte immunoglobulin-like receptor B1 (LILR1/ILT2/CD85j),LILRB2 (ILT4/CD85d) and KIR2DL4 (or CD158d), schematized in FIG. 1A. ForLILRB receptors, the recognition site takes place through the α3 domainof HLA-G and it is unlikely affected by the peptide. LILRB1 is expressedby B cells, some T cells, some NK cells, and all monocytes/dendriticcells, whereas LILRB2 is myeloid specific and its expression isrestricted to monocytes/dendritic cells. KIR2DL4 is a specific receptorfor HLA-G, only expressed by the CD56bright subset of NK cells. LILRB1and LILRB2 have been shown to bind a wide range of classic HLA moleculesby the α3 domain and the B2M, for which HLA-G is the ligand of highestaffinity, whereas for KIR2DL4, HLA-G is the sole known ligand. Inaddition, it has been demonstrated that LILRB1 and LILRB2 present higheraffinity for HLA-G multimers than monomeric structures. It is importantto bring up the difference between the way LILRB1 and LILRB2 bind totheir ligands: LILRB1 shows higher affinity for HLA-G heavy chainassociated to the β2M, whereas LILRB2 shows remarkably distinctMHCI-binding recognition by binding more the α3 domain than β2M,involving the aromatic amino acids Phe-195 and Tyr-197. This explainsthe β2M independent HLA-G binding of the latter receptor and its higheraffinity for β2M free isoforms.

By linking these receptors, HLA-G acts as a down-regulator of the immunesystem for which some of the functions had been described: inhibition ofthe cytolytic function of uterine and peripheral blood NK cells, theantigen-specific cytolytic function of cytotoxic T lymphocytes, thealloproliferative response of CD4⁺ T cells, the proliferation of T cellsand peripheral blood NK cells, and the maturation and function ofdendritic cells, shown in FIG. 1C. Furthermore, HLA-G can induce thegeneration of suppressive cells. But, unlike classic HLA class Imolecules, no stimulatory functions had been reported to date for HLA-G,neither responses directed against allogeneic HLA-G.

HLA-G can inhibit all the immune cell subsets; thus, it can block allthe stages of the anti-tumor response. This molecule is expressed inmany types of primary tumors, metastases and malignant effusions, and itcan also be found on tumor cells and tumor-infiltrating cells. It wasshown that HLA-G expression by tumor cell lines protects them fromdestruction by cytotoxic T lymphocytes and NK cells. Thus, theexpression of HLA-G by malignant cells may prevent tumor immuneelimination by inhibiting the activity of tumor infiltrating NK,cytotoxic T lymphocytes (CTL) and antigen presenting cells (APCs).

HLA-G expression is mainly controlled at the transcriptional level by aunique gene promoter and at the post-transcriptional level byalternative splicing, mRNA stability, translation and protein transportto the cell surface.

The primary transcript of HLA-G is alternatively spliced resulting inthe expression of seven isoforms, where four are membrane-bound (HLA-G1,HLA-G2, HLA-G3 and HLA-G4) and three are soluble (HLA-G5, HLA-G6 andHLA-G7). HLA-G1 and HLA-G5 present the typical structure of a classicalHLA class I molecule: a heavy chain constituted of three globulardomains non-covalently bound to β2-microglobulin (β2M) and a peptide,while the other isoforms are shorter, lacking one or two domains of theheavy chain, and should not bind β2M (FIG. 1A).

HLA-G1 and HLA-G5 are considered the most abundant isoforms, probablybecause of the lack of antibodies diversity against other isoforms,particularly the lack of antibodies against β2M free isoforms. HLA-G1isoform is the complete isoform with α1, α2 and α3 domains associatedwith β2-microglobulin. The HLA-G2 isoform has no α2 domain, while HLA-G3has no α2 and α3 domains, and HLA-G4 has no α3 domain. None of theisoforms HLA-G2, HLA-G3 and HLA-G4 binds β2M. The soluble HLA-G5 andHLA-G6 isoforms contain the same extra globular domains than HLA-G1 andHLA-G2, respectively. The HLA-G7 isoform has only the al domain linkedto two amino acids encoded by intron 2. HLA-G5 isoform binds β2M whilethe isoforms HLA-G6 and HLA-G7 do not bind β2M.

In addition, HLA-G molecules can form dimers through the creation ofdisulfide bonds between two unique cysteine residues at positions 42(Cys42-Cys42 bonds) and 147 (Cys42-Cys147 bonds) of the HLA-G heavychain. The dimerization has an oblique orientation that exposes theHLA-G receptor binding sites of the α3 domain upwards, making them moreaccessible to the receptors. Consequently, HLA-G dimers bind receptorswith higher affinity and slower dissociation rates than monomers, andsignal more efficiently than monomers as well.

Alternative names are HLA-G histocompatibility antigen class I or G orMHC-G. HLA-G is described in databases under the following accessionnumbers: Gene ID: 3135, UniGene Hs.512152. This protein is disclosed inUniProt under accession number: P17693. The GenBank entry of thesequence of the protein and mRNA are respectively NP_002118.1. andNM_002127.5.

When comparing HLA-G isoforms sequences, HLA-G1 is generally chosen asthe canonical sequence, i.e. the sequence of DNA, RNA, or amino acidsthat reflects the most frequent nucleic acid or base or amino acid ateach position, which is why database generally refer to this isoformsequence under the name “HLA-G”. HLA-G2 to G7 differ from HLA-G1 byamino acid deletion(s) and/or substitution(s). HLA-G human isoforms aredescribed under the Uniprot accession number P17693-1 for HLA-G1,P17693-2 for HLA-G2, P17693-3 for HLA-G3, P17693-4 for HLA-G4, P17693-5for HLA-G5, P17693-6 for HLA-G6, P17693-7 for HLA-G7.

Antibodies Against HLA-G Isoforms

The present invention provides an antibody that specifically binds oneto six, preferably two to five HLA-G isoform(s) among the seven HLA-Gisoforms, but does not specifically bind or recognize all the HLA-Gisoforms. For instance, the antibody can specifically bind one, two,three, four, five or six HLA-G isoforms. The HLA-G isoforms can beselected from the group consisting of HLA-G1, HLA-G2, HLA-G3, HLA-G4,HLA-G5, HLA-G6 and HLA-G7, preferably from HLA-G1, HLA-G2, HLA-G5 andHLA-G6.

For example, the antibody or the antigen binding domain of the CARaccording to the invention can recognize:

-   -   HLA-G1, HLA-G4 and HLA-G5, if the epitope recognized by the        antibody or antigen binding domain is on the α2 domain of HLA-G,    -   HLA-G1, HLA-G2, HLA-G5 and HLA-G6, if the epitope recognized by        the antibody or antigen binding domain is on the α3 domain of        HLA-G,    -   HLA-G1 and HLA-G5, if the epitope recognized by the antibody or        antigen binding domain is on the β2M domain of HLA-G or on a        domain which is specific of the HLA-G associated with the β2M        domain.

In a particular embodiment the antibody or fragment thereof does notrecognize all the HLA-G isoforms, i.e. the antibody or the antigenbinding domain of the CAR according to the invention does not recognizean epitope of the α1 domain of HLA-G.

In one aspect, the antibody can specifically bind HLA-G1 and HLA-G5isoforms. Then, the antibody does not substantially bind the other HLA-Gisoforms, especially HLA-G2, HLA-G3, HLA-G4, HLA-G6 and HLA-G7. Morespecifically, the antibody is specific of the HLA-G isoforms associatedwith β2M. In this context, the antibody does not substantially bindHLA-G1 and HLA-G5 isoforms devoid of β2M.

It is provided herein an antibody 15E7. In particular, 15E7 is a scFvantibody having a heavy chain sequence as disclosed in SEQ ID NO: 3 anda light chain sequence as disclosed in SEQ ID NO: 4. The CDRs of theantibody 15E7 have the following sequences, according to Kabat:

-   -   Heavy chain CDR1 of SEQ ID NO: 11;    -   Heavy chain CDR2 of SEQ ID NO: 12,    -   Heavy chain CDR3 of SEQ ID NO: 13,    -   Light chain CDR1 of SEQ ID NO: 14,    -   Light chain CDR2 of SEQ ID NO: 15, and    -   Light chain CDR3 of SEQ ID NO: 16.    -   Accordingly, the present invention relates to an antibody having

-   (a) (i) a heavy chain comprising CDR 1, 2 and 3 of the heavy chain    variable region of SEQ ID NO: 3 and (ii) a light chain comprising    CDR 1, 2 and 3 of the light chain variable region of SEQ ID NO: 4;

-   (b) (i) a heavy chain comprising CDR 1, 2 and 3 (HCDR1, HCDR2,    HCDR3) comprising a sequence of SEQ ID NO: 11, 12 and 13,    respectively, and (ii) a light chain comprising CDR 1, 2 and 3    (LCDR1, LCDR2, LCDR3) comprising a sequence of SEQ ID NO: 14, 15 and    16, respectively, optionally wherein each CDR may optionally    comprise 1, 2, 3 or 4 amino acid substitutions, deletions or    insertions; or

-   (c) a heavy chain variable region and a light chain variable region    of SEQ ID NOS: 3 and 4 or a heavy chain variable region having at    least 80, 85, 90 or 95% of identity with SEQ ID NOS: 3 and a light    chain variable region having at least 80, 85, 90 or 95% of identity    with SEQ ID NO: 4.

Said antibody can be a chimeric, human or humanized. Said antibody canbe an antibody fragment selected from Fab, Fab′, Fab′-SH, F(ab′) 2, Fv,a diabody, a single-chain antibody fragment, or a multispecific antibodycomprising multiple different antibody fragments. Said antibody can beconjugated or covalently bound to a toxic agent or to a detectablelabel.

In another aspect, the antibody can specifically bind HLA-G2 and HLA-G6isoforms. Then, the antibody does not substantially bind the other HLA-Gisoforms, especially isoforms associated with the β2M subunit such asHLA-G1, HLA-G3, HLA-G4, HLA-G5 and HLA-G7. More specifically, theantibody is specific of the β2M-free HLA-G isoforms and specificallybinds both HLA-G2 and HLA-G6 and/or both HLA-G1-β2M free and HLA-G5-β2Mfree isoforms.

It is provided herein an antibody LFTT-1. In particular, LFTT-1 is ascFv antibody having a heavy chain sequence as disclosed in SEQ ID NO: 1and a light chain sequence as disclosed in SEQ ID NO: 2. The CDRs of theantibody LFTT-1 have the following sequences, according to Kabat:

-   -   Heavy chain CDR1 of SEQ ID NO: 5;    -   Heavy chain CDR2 of SEQ ID NO: 6,    -   Heavy chain CDR3 of SEQ ID NO: 7,    -   Light chain CDR1 of SEQ ID NO: 8,    -   Light chain CDR2 of SEQ ID NO: 9, and    -   Light chain CDR3 of SEQ ID NO: 10.

Accordingly, the present invention relates to an antibody having

-   (a) (i) a heavy chain comprising CDR 1, 2 and 3 of the heavy chain    variable region of SEQ ID NO: 1 and (ii) a light chain comprising    CDR 1, 2 and 3 of the light chain variable region of SEQ ID NO: 2;-   (b) (i) a heavy chain comprising CDR 1, 2 and 3 (HCDR1, HCDR2,    HCDR3) comprising a sequence of SEQ ID NO: 5, 6 and 7, respectively,    and (ii) a light chain comprising CDR 1, 2 and 3 (LCDR1, LCDR2,    LCDR3) comprising a sequence of SEQ ID NO: 8, 9 and 10,    respectively, optionally wherein each CDR may optionally comprise 1,    2, 3 or 4 amino acid substitutions, deletions or insertions; or-   (c) a heavy chain variable region and a light chain variable region    of SEQ ID NOS: 1 and 2 or a heavy chain variable region having at    least 80, 85, 90 or 95% of identity with SEQ ID NOS: 1 and a light    chain variable region having at least 80, 85, 90 or 95% of identity    with SEQ ID NO: 2.

Said antibody can be a chimeric, human or humanized. Said antibody canbe an antibody fragment selected from Fab, Fab′, Fab′-SH, F(ab′) 2, Fv,a diabody, a single-chain antibody fragment, or a multispecific antibodycomprising multiple different antibody fragments. Said antibody can beconjugated or covalently bound to a toxic agent or to a detectablelabel.

In a particular embodiment, the antibody is a multispecific antibody,preferably a bispecific antibody. Bispecific antibodies comprise twodifferent F(ab) fragments that recognize two different epitopes eitheron the same or on different antigens. Particularly, the bispecificantibody according to the invention binds different epitopes on the sameHLA-G isoform(s) or different HLA-G isoforms. Preferably, the bispecificantibody specifically binds different HLA-G isoforms and comprises aF(ab) that recognizes HLA-G/β2M free isoforms and a second F(ab) thatrecognizes HLA-G isoforms associated with the β2M domain.

In one embodiment, the bispecific antibody comprises one F(ab) thatspecifically binds to HLA-G isoforms that are free of β2M, preferably toboth HLA-G2 and HLA-G6 and/or to both HLA-G1/β2M free and HLA-G5/β2Mfree isoforms, and one other F(ab) that specifically binds to HLA-Gisoforms associated with β2M, preferably HLA-G1 and HLA-G5 isoforms. Inthis particular embodiment, the bispecific antibody comprises one F(ab)from the LFTT-1 antibody with the CDRs described hereabove (SEQ ID No.5-10) and one F(ab) of the 15E7 antibody with the CDRs describedhereabove (SEQ ID No. 11-16).

In another embodiment, the bispecific anti-HLA-G antibody comprises acontinuous heavy chain constructed of an Fc (Hinge-CH2-CH3) followed bya first antibody (antibody 1) Fab heavy chain (CH1-VH) and thesuccessive Fab heavy chain (CH1-VH) of a second antibody (antibody 2),the latter joined by a polypeptide linker sequence. During proteinexpression the resulting heavy chain assembles into dimers while theco-expressed antibody 1 and antibody 2 light chains (VL-CL) associatewith their cognate heavy chains to form the final tandem F(ab)′2-Fcmolecule. Preferably, the antibody 1 and the antibody 2 are different,preferably are LFTT-1 antibody and 15E7 antibody respectively.

In one embodiment, the antibody or fragment thereof does not bind orrecognize all of the HLA-G isoforms. Particularly, the antibody orfragment thereof that specifically binds to one or several but not allHLA-G isoform(s) has a Kd affinity constant of at least 10⁻⁶ M. Incertain aspects, the antibody or fragment thereof binds one or severalbut not all HLA-G isoform(s) with high affinities of at least about 10⁻⁷M, and preferably at least about 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M.

In a particular embodiment, the antibody or fragment thereof does notbind or recognize the alpha1 domain of HLA-G isoforms. This means thatsuch antibody or fragment thereof can bind or recognize HLA-G isoformslacking of alpha-1 domain, for example HLA-G isoforms such as describedin Tronik-Le Roux et al., Molecular Oncology 11 (2017) 1561-1578, thatcontain the alpha2 and alpha3 domains or only the alpha3 domain. Forexample, such antibody or fragment thereof can bind the alpha2, alpha3or β2M domain.

The sequence of the antibody or antibody fragment according to theinvention may be used in a method to prepare a CAR or to prepare apharmaceutical composition. Alternatively, the antibody or antibodyfragment according to the invention may be used to detect HLA-Gisoform(s) in diagnosis tests such as immunoassays.

Antibodies or antibody fragments can be identified, for example, byimmunoassays such as radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs) and Surface Plasmon Resonance (SPR) assaysor other techniques known to those of skill in the art.

Preferably, antibodies (including antibody fragments thereof) thatspecifically bind to one or several HLA-G isoform(s) do notsignificantly cross-react with other antigens (i.e., is not detectablein routine immunological assays). An antibody binds specifically to anantigen when it binds to the antigen with higher affinity than to anycross-reactive antigen as determined using experimental techniques, suchas Western blot (WB), radioimmunoassays (RIAs) and enzyme-linkedimmunosorbent assays (ELISAs), particularly competitive ELISA.

Chimeric Antigen Receptors (CARs) Against HLA-G Isoforms

A CAR typically comprises an ectodomain (extracellular domain) and anendodomain (cytoplasmic domain), preferably joined by a hinge domain anda transmembrane domain. Particularly, the CAR according to the inventionoptionally comprises a cleavable linker and a reporter. The ectodomain,expressed on the surface of the cell, comprises an antigen bindingdomain or receptor domain, optionally a signal peptide that directs theantigen binding domain into the endoplasmic reticulum for processing.The extracellular domain comprises an antigen binding domain thatspecifically recognizes a target antigen. As a non-limiting example, theantigen binding domain can be an antibody, preferably a single chainantibody, such as a scFv. The spacer region links the antigen bindingdomain to the transmembrane domain and is designed to be sufficientlyflexible to allow the antigen binding domain to orient in a manner thatallows antigen recognition. The transmembrane domain is typically ahydrophobic alpha helix, typically, that spans across the lipid bilayerof the cell membrane. The endodomain of the CAR is composed of a signaltransmitting peptide that spreads an activation signal intracellularlyto the cell cytoplasm, thereby stimulating the cell expressing the CAR.The endodomain may include several signaling domains, as explained videinfra.

In some embodiments, a CAR comprises at least an extracellular antigenbinding domain, a transmembrane domain and a cytoplasmic signalingdomain comprising a functional signaling domain derived from astimulatory molecule and/or costimulatory molecule as defined below. Insome embodiments, the set of polypeptides include a dimerization switchthat, upon the presence of a dimerization molecule, can couple thepolypeptides to one another, e.g., can couple an antigen binding domainto an intracellular signaling domain. In one aspect, the cytoplasmicsignaling domain further comprises one or more functional signalingdomains derived from at least one costimulatory molecule as definedbelow.

In one aspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, a transmembrane domain and anintracellular signaling domain comprising a functional signaling domainderived from a stimulatory molecule. In one aspect, the CAR comprises achimeric fusion protein comprising an extracellular antigen bindingdomain, a transmembrane domain and an intracellular signaling domaincomprising a functional signaling domain derived from a costimulatorymolecule and a functional signaling domain derived from a stimulatorymolecule.

Preferably, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, optionally a hinge domain, atransmembrane domain and an intracellular signaling domain comprisingtwo functional signaling domains derived from one or more costimulatorymolecule(s) and a functional signaling domain derived from a stimulatorymolecule. In one aspect the CAR comprises an optional leader sequence orpeptide signal sequence at the amino-terminus (N-ter) of the CAR fusionprotein.

In a particular embodiment, the CAR further comprises a cleavable linkerand a reporter, to facilitate identification and selection of expressingcells from the population of cells sought to be transfected with the CARaccording to the invention, preferably sequentially at thecarboxy-terminus (C-ter) of the CAR fusion protein. According to theinvention, the engineered CAR has one, two, three, four, five, six,seven, eight or more components, and in some embodiments at least onecomponent facilitates targeting or binding of the immune cell tospecific HLA-G isoform(s). Preferably, this at least one component isderived from a monoclonal antibody as defined in the previous section.Preferably, this component is a scFv.

Preferably, the anti-HLA-G CAR sequentially comprises or consists in,from N to C terminus: optionally a peptide signal sequence, ananti-HLA-G antibody or fragment thereof, preferably an anti-HLA-G scFv,a spacer domain, a transmembrane domain, at least one intracellulardomain, a cleavable linker and a truncated human CD19 reporter. All ofthese components will be more specifically described herebelow.

Particularly, the CAR according to the invention, particularly theantigen binding domain, the antibody or the scFv according to theinvention, may specifically bind one to six, preferably two to five,HLA-G isoform(s), more preferably to one or two HLA-G isoform(s) or twoor three HLA-G isoforms, but does not bind to all HLA-G isoforms. Forinstance, the CAR can specifically bind one, two, three, four, five orsix HLA-G isoforms. The HLA-G isoforms can be selected from the groupconsisting of HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6 and HLA-G7,preferably from HLA-G1, HLA-G2, HLA-G5 and HLA-G6.

In one aspect, the CAR can specifically bind β2M-associated HLA-Gisoforms, such as HLA-G1 and HLA-G5 isoforms. Then, the CAR does notsubstantially bind the other HLA-G isoforms, especially HLA-G2, HLA-G3,HLA-G4, HLA-G6 and HLA-G7. More specifically, the CAR is specific of theHLA-G isoforms associated with β2M. In this context, the CAR does notsubstantially bind HLA-G1 and HLA-G5 isoforms devoid of β2M.

In another aspect, the CAR can specifically bind HLA-G2 and HLA-G6isoforms. Then, the CAR does not substantially bind the other HLA-Gisoforms, especially HLA-G1, HLA-G3, HLA-G4, HLA-G5 and HLA-G7. Morespecifically, the CAR is specific of the β2M-free HLA-G isoforms and canspecifically bind to both HLA-G2 and HLA-G6 and/or to both HLA-G1/β2Mfree and HLA-G5/β2M free isoforms.

Antigen Binding Domain

The external domain of the CAR according to the invention is an antigenbinding domain. Particularly, this antigen binding domain is derivedfrom the antibody as defined in the above section.

Antigen targeting or antigen recognition by CAR molecules most commonlyinvolves the use of a single chain variable fragment (scFv) that hasbeen assembled from a monoclonal antibody. However, alternativetargeting moieties include ligands (Altenschmidt et al. (1996) Clin.Cancer Res. 2: 1001-8; Muniappan, et al. (2000) Cancer Gene Ther. 7:128-134), peptides (Pameijer et al. (2007) Cancer Gene Ther. 14:91-97),chimeric ligands (Davies et al. (2012) Mol. Med. 18:565-576), receptorderivatives (Zhang et al. (2012) J Immunol. 189:2290-9), and singledomain antibodies (Sharifzadeh et al. (2012) Cancer Res. 72: 1844-52).Any desired antibody or antibody fragment thereof that specificallyrecognizes and binds a target antigen, in particular HLA-G isoforms asdefined above, may be incorporated in a CAR according to the invention.

In one embodiment, such antigen binding domain is an antibody,preferably a single chain antibody. Preferably, the antibody is ahumanized antibody or a non-humanized murine antibody. Particularly,such antigen binding domain is an antibody fragment selected fromfragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chainantibody fragments, single chain variable fragments (scFv), singledomain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, andmulti-specific antibodies formed from antibody fragments. In particularembodiments, the antibodies are single-chain antibody fragmentscomprising a variable heavy chain region and/or a variable light chainregion, such as scFv. Particularly, such antigen binding domain isselected from a Fab and a scFv.

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells. In some embodiments, theantibodies are recombinantly-produced fragments, such as fragmentscomprising arrangements that do not occur naturally, such as those withtwo or more antibody regions or chains joined by synthetic linkers,e.g., peptide linkers, and/or that may not be produced by enzymedigestion of a naturally-occurring intact antibody. In some aspects, theantibody fragments are scFvs.

In embodiments wherein the antigen targeting domain is a scFv, the scFvcan be derived from the variable heavy chain (VH) and variable lightchain (VL) regions of an antigen-specific mAb linked by a flexiblelinker. The scFv retains the same specificity and a similar affinity asthe full antibody from which it was derived (Muniappan et al. (2000)Cancer Gene Ther. 7: 128-134). Various methods for preparing an scFv canbe used including methods described in U.S. Pat. No. 4,694,778; Bird etal. (1988) Science 242:423-442; Ward et al. (1989) Nature 334:54454; andSkerra et al. (1988) Science 242: 1038-1041. In certain embodiments, thescFv may be a humanized or is a fully human scFv.

The antigen binding domain that specifically binds to a particularisoform of HLA-G may be cross-reactive with related antigens, forexample with one to five, preferably two to four other different HLA-Gisoform(s).

In some aspects, the antigen binding domain may be derived from anantibody or fragment thereof that has one or more specified functionalfeatures, such as binding properties, including binding to particularepitopes, such as epitopes that are similar to or overlap with those ofother antibodies, the ability to compete for binding with otherantibodies, and/or particular binding affinities. In some embodiments,the antigen binding domain, the CARs comprising such, and the cellscomprising such CARs display a binding preference for targetantigen-expressing cells as compared to target antigen-negative cells.In some embodiments, the binding preference is observed where asignificantly greater degree of binding is measured to theantigen-expressing, as compared to the non-expressing cells. In somecases, the total degree of observed binding to the target antigen or tothe antigen-expressing cells is approximately the same, at least asgreat or greater than that observed for non-antigen specific domains,CARs, or cells. In any of the provided embodiments, comparison ofbinding properties, such as affinities or competition, may be viameasurement by assays known in the art such as mentioned above.

In another embodiment, the antigen binding domain of the CARspecifically binds to one or several HLA-G isoform(s) with a Kd affinityconstant of at least 10⁻⁶ M. In certain aspects, the antigen bindingdomain of the CAR binds one or several HLA-G isoform(s) with highaffinities of at least about 10⁻⁷ M, and preferably at least about 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M. Preferably, the antigen binding domain of the CARbinds one or several HLA-G isoform(s) with an affinity of about 10⁻⁹ M.

In one aspect, the antigen binding fragment of the CAR can specificallybind to both HLA-G2 and HLA-G6 and/or to both HLA-G1/β2M free and toHLA-G5/β2M free isoforms. Then, the antigen binding fragment of the CARdoes not substantially bind the other HLA-G isoforms, especially HLA-G1and HLA-G5 β2M-associated isoforms, HLA-G3, HLA-G4, and HLA-G7. Morespecifically, the antigen binding fragment of the CAR is specific of theβ2M-free HLA-G isoforms and does not substantially bind β2M-associatedHLA-G isoforms.

In a particular embodiment, the antigen binding fragment of the CARcomprises i) a heavy chain variable region that comprises SEQ ID NO: 1or a sequence having at least 80, 85, 90 or 95% of identity with SEQ IDNO: 1; and/or

(ii) the light chain variable region comprises SEQ ID NO: 2 or asequence having at least 80, 85, 90 or 95% of identity with SEQ ID NO:2.

In an additional particular embodiment, the antigen binding fragment ofthe CAR comprises (i) a heavy chain comprising CDR 1, 2 and 3 of theheavy chain variable region of SEQ ID NO: 1 and (ii) a light chaincomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 2.

In a further particular embodiment, the antigen binding fragment of theCAR comprises (i) a heavy chain comprising CDR 1, 2 and 3 (HCDR1, HCDR2,HCDR3) comprising a sequence of SEQ ID NO: 5, 6 and 7, respectively, and(ii) a light chain comprising CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3)comprising a sequence of SEQ ID NO: 8, 9 and 10, respectively,optionally wherein each CDR may optionally comprise 1, 2, 3 or 4 aminoacid substitutions, deletions or insertions.

In another aspect, the antigen binding fragment of the CAR canspecifically bind the HLA-G isoforms associated with β2M such as HLA-G1and HLA-G5 isoforms. Then, the antigen binding fragment of the CAR doesnot substantially bind the other HLA-G isoforms, especially β2M freeisoforms such as HLA-G2, HLA-G3, HLA-G4, HLA-G6 and HLA-G7. In thiscontext, the antigen binding fragment of the CAR does not substantiallybind HLA-G1 and HLA-G5 isoforms devoid of β2M.

In a particular embodiment, the antigen binding fragment of the CARcomprises i) a heavy chain variable region that comprises SEQ ID NO: 3or a sequence having at least 80, 85, 90 or 95% of identity with SEQ IDNO: 3; and/or

(ii) the light chain variable region comprises SEQ ID NO: 4 or asequence having at least 80, 85, 90 or 95% of identity with SEQ ID NO:4.

In an additional particular embodiment, the antigen binding fragment ofthe CAR comprises (i) a heavy chain comprising CDR 1, 2 and 3 of theheavy chain variable region of SEQ ID NO: 3 and (ii) a light chaincomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 4.

In a further particular embodiment, the antigen binding fragment of theCAR comprises (i) a heavy chain comprising CDR 1, 2 and 3 (HCDR1, HCDR2,HCDR3) comprising a sequence of SEQ ID NO: 11, 12 and 13, respectively,and (ii) a light chain comprising CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3)comprising a sequence of SEQ ID NO: 14, 15 and 16, respectively,optionally wherein each CDR may optionally comprise 1, 2, 3 or 4 aminoacid substitutions, deletions or insertions.

Particularly, the antigen binding fragment of the CAR does not bind orrecognize all of the HLA-G isoforms. In one embodiment, the antigenbinding fragment of the CAR does not bind or recognize the alpha1 domainof HLA-G isoforms. This means that such antigen binding fragment canrecognize HLA-G isoforms lacking of alpha-1 domain, for example HLA-Gisoforms such as described in Tronik-Le Roux et al., Molecular Oncology11 (2017) 1561-1578 that contain the alpha2 and alpha3 domains or onlythe alpha3 domain. For example, such antigen binding fragment can bindthe alpha2, alpha3 or β2M domain.

In a particular embodiment, the antigen binding domain is amultispecific antigen binding domain, preferably a bispecific antigenbinding domain. For instance, the bispecific antigen binding fragmentcomprises a domain that recognizes HLA-G/β2M free isoforms and a seconddomain that recognizes HLA-G isoforms associated with the β2M domain.Even more preferably, the bispecific antigen binding domain comprisesone domain that specifically binds to both HLA-G2 and HLA-G6 and/or toboth HLA-G1/β2M free and HLA-G5/β2M free isoforms, and another domainthat specifically binds to HLA-G1 and HLA-G5 isoforms associated withthe β2M subunit. Preferably, the bispecific antigen binding fragment isderived from both LFTT-1 antibody with the CDRs described hereabove (SEQID No. 5-10) and the 15E7 antibody with the CDRs described hereabove(SEQ ID No. 11-16).

In a particular embodiment, the antigen binding fragment of the CAR is abispecific antigen binding domain that comprises (i) a domain comprisingCDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) comprising a sequence of SEQ ID NO:11, 12 and 13, respectively, (ii) a domain comprising CDR 1, 2 and 3(LCDR1, LCDR2, LCDR3) comprising a sequence of SEQ ID NO: 14, 15 and 16,respectively, (iii) a domain comprising CDR 1, 2 and 3 (HCDR1, HCDR2,HCDR3) comprising a sequence of SEQ ID NO: 5, 6 and 7, respectively, and(iv) a domain comprising CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) comprisinga sequence of SEQ ID NO: 8, 9 and 10, respectively, optionally whereineach CDR may optionally comprise 1, 2, 3 or 4 amino acid substitutions,deletions or insertions.

Such bispecific antigen binding domain suitable for a CAR can forexample be tandem-scFv or nanobodies (De Munter et al., Molecularsciences 2018).

In some embodiments, the antigen binding domain comprises a scFvcomprising the CDR sequences of an anti-HLA-G monoclonal antibody. CDRsmay be determined using conventional methods. The precise amino acidsequence boundaries of a given CDR or FR can be readily determined usingany of a number of well-known schemes, including those described byKabat et al. (1991), “Sequences of Proteins of Immunological Interest,”5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis andbinding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact”numbering scheme), Lefranc M P et al, “IMGT unique numbering forimmunoglobulin and T cell receptor variable domains and Ig superfamilyV-like domains,” Dev Comp Immunol, 2003 January; 27(I):55-77 (“IMGT”numbering scheme), and Honegger A and Pluckthun A, “Yet anothernumbering scheme for immunoglobulin variable domains: an automaticmodeling and analysis tool,” J Mol Biot, 2001 Jun. 8; 309(3):657-70,(“Aho” numbering scheme). In specific embodiments, the antigen bindingdomain is a single-chain variable fragment (scFv). The scFv may alsocomprise a peptide linker. The peptide linker connecting scFv V_(H) andV_(L) domains joins the carboxyl terminus of one variable region domainto the amino terminus of the other variable domain without compromisingthe fidelity of the V_(H)-V_(L) paring and antigen-binding sites.Peptide linkers can vary from 10 to 30 amino acids in length. In oneembodiment, the scFv peptide linker is a Gly/Ser linker and comprisesone or more repeats of the amino acid sequence Gly-Gly-Gly-Ser orGly-Gly-Gly-Gly-Ser. In one embodiment, the flexible polypeptide linkerincludes, but is not limited to (Gly₃Ser)₃ and (Gly₄Ser)₃.

Transmembrane and Hinge Domain

Hinge Domain

When a T cell interacts with an antigen-presenting cell, animmunological synapse with an intermembrane distance of about 15 nm isformed. This distance is dictated by the architecture of TCR and thepeptide-MHC complex. This spatial separation is important for effectivetriggering of the phosphorylation cascade and T-cell activation. Whenartificially shortened, this protein gets a chance to stay within thesynapse, resulting in the suppression of activation signals.

Given that the position of the epitope recognized by a specific scFv onthe target cell surface is generally fixed, the length and rigidity ofthe extracellular spacer (the hinge module) in the CAR need to beadjusted to ensure maximum steric compatibility with the scFv and theformation of a compact synapse. In some aspects, a portion of theimmunoglobulin constant region serves as a spacer region between theantigen binding domain, e.g., scFv, and transmembrane domain. The spacercan be of a length that provides for increased responsiveness of thecell following antigen binding, as compared to in the absence of thespacer. The spacer domain preferably has a sequence that promotesbinding of a CAR with an antigen and enhances signaling in a cell.Examples of an amino acid that is expected to promote the bindinginclude cysteine, a charged amino acid, and serine and threonine in apotential glycosylation site, and these amino acids can be used as anamino acid constituting the spacer domain.

In some embodiments, the CAR comprises a hinge sequence between theantigen binding domain and the transmembrane domain and/or between thetransmembrane domain and the cytoplasmic domain. The hinge domain can beup to 150 amino acids, preferably 10 to 100 amino acids, even morepreferably 50 to 100 amino acids in length. One ordinarily skilled inthe art will appreciate that a hinge sequence is a short sequence ofamino acids that facilitates flexibility (see e.g., Woof et al, Nat.Rev. Immunol, 4(2): 89-99 (2004)). The hinge sequence can be anysuitable sequence derived or obtained from any suitable molecule. Insome embodiments, the length of the hinge sequence may be optimizedbased on the distance between the CAR and the binding epitope, e.g.,longer hinges may be optimal for membrane proximal epitopes.

The hinge may be derived from or include at least a portion of animmunoglobulin Fc region, for example, an IgG1 Fc region, an IgG2 Fcregion, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgMFc region, or an IgA Fc region. In certain embodiments, the hinge domainincludes at least a portion of an IgG1, an IgG2, an IgG3, an IgG4, anIgE, an IgM, or an IgA immunoglobulin Fc region that falls within itsCH2 and CH3 domains. In some embodiments, the spacer domain may alsoinclude at least a portion of a corresponding immunoglobulin hingeregion. In some embodiments, the hinge is derived from or includes atleast a portion of a modified immunoglobulin Fc region, for example, amodified IgG1 Fc region, a modified IgG2 Fc region, a modified IgG3 Fcregion, a modified IgG4 Fc region, a modified IgE Fc region, a modifiedIgM Fc region, or a modified IgA Fc region. The modified immunoglobulinFc region may have one or more mutations (e.g., point mutations,insertions, deletions, duplications) resulting in one or more amino acidsubstitutions, modifications, or deletions that cause impaired bindingof the spacer domain to an Fc receptor (FcR). In some aspects, themodified immunoglobulin Fc region may be designed with one or moremutations which result in one or more amino acid substitutions,modifications, or deletions that cause impaired binding of the spacerdomain to one or more FcR including, but not limited to, FcyRl, FcyR2A,FcyR2Bl, FcyR2B2, FcyR3A, FcyR3B, FcsRl, FcsR2, FcaRl, Fca/μK, or FcRn.

Exemplary hinges include, but are not limited to, a CD8a hinge, a CD28hinge, IgG1/IgG4 (hinge-Fc part) sequences (in single studies, CD4, CD7,and IgD) IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, orIgG4 hinge linked to the CH3 domain, those described in Hudecek et al.(2013) Clin. Cancer Res., 19:3153, international patent applicationpublication number WO2014031687, U.S. Pat. No. 8,822,647 or publishedapp. No. US2014/0271635. As hinge domain, the invention relates to allor a part of residues 118 to 178 of CD8a (GenBank Accession No.NP_001759.3), residues 135 to 195 of CD8 (Gen Bank Accession No.AAA35664), residues 315 to 396 of CD4 (GenBank Accession No.NP_000607.1), or residues 137 to 152 of CD28 (GenBank Accession No.NP_006130.1) can be used. Also, as the spacer domain, a part of aconstant region of an antibody H chain or L chain (CHI region or CLregion) can be used. Further, the spacer domain may be an artificiallysynthesized sequence. Particularly, the CAR according to the inventioncomprises a hinge selected from CD8a, CD28, and IgG1/IgG4 (hinge-Fcpart) sequences (in single studies, CD4, CD7, and IgD). This choice isbased on the fact that these sequences are relatively neutral, flexible,and have been well-characterized structurally.

Preferably, the hinge domain comprises or consists of (i) CD28 hinge,(ii) CD8 alpha hinge, (iii) a human IgG4 hinge domain, (iv) a human IgG4hinge domain and a CH3 human IgG4 domain or (v) a mutated CH2 human IgG4domain, a human IgG4 hinge domain and a CH3 human IgG4 hinge domain.Even more preferably, the hinge domain comprises or consists of (a) SEQID NO: 18, (b) SEQ ID 19, (c) SEQ ID NO: 25, (d) SEQ ID NO: 25 and SEQID NO: 27, or (e) SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, or asequence having at least 80, 85, 90 or 95% identity thereto.

In one embodiment the hinge domain comprises or consists of (i) a humanIgG4 hinge domain, (ii) a human IgG4 hinge domain and a CH3 human IgG4domain or (iii) a mutated CH2 human IgG4 domain, a human IgG4 hingedomain and a CH3 human IgG4 hinge domain. Particularly, the hinge domainsequentially comprises or consists from the N terminus to the C terminusof (i) a human IgG4 hinge domain and a CH3 human IgG4 domain or (ii) amutated CH2 human IgG4 domain, a human IgG4 hinge domain and a CH3 humanIgG4 hinge domain.

In on embodiment, the CAR according to the invention comprises a spacerdomain that comprises or consists of a human IgG4 hinge domain thatcomprises or consists of the sequence set forth in SEQ ID NO: 25 or asequence having at least 80, 85, 90, 95%, 96%, 97%, 98%, 99% of identitytherewith.

In another embodiment, the CAR according to the invention comprises aspacer domain that comprises or consists of (i) a CH3 human IgG4 domainthat comprises or consists of the sequence set forth in SEQ ID NO: 27 ora sequence having at least 80, 85, 90, 95%, 96%, 97%, 98%, 99% ofidentity therewith and (ii) a human IgG4 hinge domain that comprises orconsists of the sequence set forth in SEQ ID NO: 25 or a sequence havingat least 80, 85, 90, 95%, 96%, 97%, 98%, 99% of identity therewith.

In one embodiment, the CAR according to the invention further comprisesmutated CH2 human IgG4 domain, in which the mutation in the CH2 humanIgG4 domain consists of the mutation of the amino acids EFLG(113-116)PVAand N177Q, for example as disclosed in Watanabe et al. Oncoimmunology.2016; 5(12):e1253656. Preferably, the mutated CH2 human IgG4 domaincomprises or consists of the sequence set forth in SEQ ID NO: 26 or asequence having at least 80, 85, 90, 95%, 96%, 97%, 98%, 99% of identitytherewith.

In a preferred embodiment, the CAR according to the invention comprisesa hinge domain that sequentially comprises or consists of (i) a mutatedCH2 human IgG4 domain comprises or consists of the sequence set forth inSEQ ID NO: 26 or a sequence having at least 80, 85, 90, 95%, 96%, 97%,98%, 99% of identity therewith, (ii) a CH3 human IgG4 domain thatcomprises or consists of the sequence set forth in SEQ ID NO: 27 or asequence having at least 80, 85, 90, 95%, 96%, 97%, 98%, 99% of identitytherewith and (iii) a human IgG4 hinge domain that comprises or consistsof the sequence set forth in SEQ ID NO: 25 or a sequence having at least80, 85, 90, 95%, 96%, 97%, 98%, 99% of identity therewith.

Particularly, the CAR according to the invention comprises a CD8a hingedomain, preferably comprising or consisting in the sequence of SEQ IDNO: 18 or a sequence having at least 80, 85, 90 or 95% of identitytherewith.

Another particular CAR according to the invention comprises a CD28 hingedomain, preferably comprising or consisting in the sequence of SEQ IDNO: 19 or a sequence having at least 80, 85, 90 or 95% of identitytherewith.

Transmembrane Domain

The transmembrane module functions to anchor the receptor on the cellsurface. With respect to the transmembrane domain, the CAR can bedesigned to comprise a transmembrane domain that is fused to the antigenbinding domain of the CAR. Particularly, the CAR can be designed tocomprise a transmembrane domain that is fused both to the antigenbinding domain and the endodomain of the CAR. In one embodiment, thetransmembrane domain that naturally is associated with one of thedomains in the CAR is used. In some instances, the transmembrane domaincan be selected or modified by amino acid substitution to avoid bindingof such domains to the transmembrane domains of the same or differentsurface membrane proteins to minimize interactions with other members ofthe receptor complex. The transmembrane domain may be derived eitherfrom a natural or from a synthetic source. Where the source is natural,the domain may be derived from any membrane-bound or transmembraneprotein. Typically, the transmembrane domain denotes a singletransmembrane helix of a transmembrane protein, also known as anintegral protein. This domain usually includes the transmembranesequences of CD3ζ, CD28, CD8, FcRlγ and less frequently, of CD4, CD7,OX40, and MHC (H2-Kb), the choice depending on the neighboring spacerand intracellular sequences. The transmembrane modules based on CD3ζ andFcRlγ ensure efficient incorporation of CAR into endogenous TCR. Forexample, the CAR of WO 2008/045437 describes a transmembrane portionderived from human CD8 alpha or CD28, and particularly chimeric T cellreceptor proteins with an unmodified CD8 hinge region with amino acidpositions 135 to 205, 135 to 203 or 135 to 182 (according to the aminoacid numbering of UniProtKB/Swiss-Prot P01732), each comprising cysteineresidues in positions 164 and 181. WO 95/30014 uses the unmodifiedmurine CD8 hinge region with amino acid positions 132 to 191 (accordingto the amino acid numbering of UniProtKB/Swiss-Prot P01731), comprisinga cysteine residue in position 178. In particular, US 2008/0260738 usesmodified CD8 hinge regions with amino acid positions 131 to 170 or 136to 169 (according to the amino acid numbering of UniProtKB/Swiss-ProtP01732), wherein the cysteine in position 164 is substituted withserine. Therefore, it would be known by the person skilled in the art,which transmembrane domain to choose according to the CAR domainscharacteristics.

Transmembrane regions of particular use in this invention may be derivedfrom (i.e. comprise at least the transmembrane region(s) of) CD28, CD3ε,CD4, CDS, CD8, CD9, CD 16, CD22, CD33, CD37, CD45, CD64, CD80, CD86,CD134, CD137, CD154, TCRα, TCRβ, H2-Kb, FcsR1γ, GITR or CD3ζ and/ortransmembrane regions containing functional variants thereof such asthose retaining a substantial portion of the structural, e.g.,transmembrane, properties thereof can be used. See e.g., Kahlon et al.(2004) Cancer Res. 64:9160-9166; Schambach et al. (2009) Methods Mol.Biol. 506: 191-205; Jensen et al. (1998) Biol. Blood Marrow Transplant4:75-83; Patel et al. (1999) Gene Ther. 6:412; Song et al. (2012) Blood119:696-706; Carpenito et al. (2009) Proc. Natl. Acad. Sci. USA106:3360-5; Hombach et al. (2012) Oncoimmunology 1:458-66) and Geiger etal. (2001) Blood 98:2364-71.

Alternatively, the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain. Atransmembrane domain of the invention is thermodynamically stable in amembrane. It may be a single alpha helix, a transmembrane beta barrel, abeta-helix of gramicidin A, or any other structure.

Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the intracellular signaling domain(s) of theCAR. A glycine-serine doublet may provide a suitable linker.

In one embodiment, the transmembrane domain of the CAR is selected fromCD3ζ, CD28, CD8, FcRlγ CD4, CD7, OX40, and MHC (H2-Kb) transmembranedomain. Preferably, the transmembrane domain of the CAR according to theinvention is selected from CD8 and CD28 transmembrane domain. Even morepreferably the transmembrane domain of the CAR according to theinvention is a CD28 transmembrane domain, preferably comprising orconsisting in the sequence of SEQ ID NO: 20 or a sequence having atleast 80, 85, 90 or 95% of identity therewith.

Intracellular Domain

In certain embodiments, a cytoplasmic or intracellular signaling domain,such as those derived from the T cell receptor ζ-chain, is employed asat least part of the chimeric receptor in order to produce stimulatorysignals for T lymphocyte proliferation and effector function followingengagement of the chimeric receptor with the target antigen.

The role of the signaling module of CARs is to transduce the activationsignal to an immune cell as soon as the extracellular domain hasrecognized the antigen. In T cells, activation begins with thephosphorylation of immunoreceptor tyrosine-based activation motif(ITAMs) in the cytoplasmic portion of the CD3ζ subunit of the TCRcomplex. Thus, in most CAR designs implemented to date, signalingsequences from CD3ζ are used as a module that triggers cell lyticactivity.

First-generation CARs, which contained the CD3ζ chain only, sentexclusively activation signal to the cell. This led to a cytotoxicreaction against tumor cells but did not provide enhanced proliferationof activated CAR T cells. In principle, the proliferation signal couldpotentially be provided by the native co-receptors present on the CAR Tcells; however, many tumors do not express the corresponding ligands.Thus, the second-generation CARs comprise a cytoplasmic domainadditionally containing the costimulatory CD28 domain, fused togetherwith CD3ζ, to overcome this difficulty. This CAR design provides bothactivation and proliferation signal to the T cell; as a result, the cellis activated, destroys the target cell, and proliferates. Besides CD28,signaling sequences from costimulatory receptors, such as CD134(TNFRSF4, OX40), CD154 (CD40L), CD137 (4-1BB), ICOS (CD278), CD27, CD244(2B4), were successfully tested in CARs.

The third generation of CARs is based on combining two or morecostimulatory sequences (such as 4-1BB-CD28-CD3ζ. These receptorssecrete a broader range of cytokines (including TNFα, GM-CSF, and IFNγ),are less susceptible to activation-induced cell death, and show higherefficacy in tumor elimination in mouse models. One or multipleendodomains may be employed, as so-called third generation CARs have atleast 2 or 3 signaling domains fused together for additive orsynergistic effect, for example.

The CAR of the invention may be a first generation, a second generation,or a third generation CAR as described hereabove. Preferably, the CAR isa second or third generation CAR. Even more preferably, the CAR is athird generation CAR when expressed by a T cell and a first generationCAR when expressed by a NK ora NKT cell.

In some embodiments, the intracellular signaling endodomain transmits asignal into a cell when the extracellular antigen targeting domainpresent within the same molecule binds to an antigen.

T cell activation is transmitted by two different kinds of cytoplasmicsignaling endodomains, that is, a sequence for initiatingantigen-dependent primary activation via a TCR complex (primarycytoplasmic signaling endodomain) and a sequence for actingantigen-independently to provide a secondary or costimulatory signal(secondary cytoplasmic signaling endodomain or costimulatoryendodomain). Therefore, while some embodiments embrace a CAR with only aprimary cytoplasmic signaling endodomain, in other embodiments, a CAR ofthe invention includes a primary signaling endodomain and a secondarycytoplasmic signaling endodomain.

The intracellular signaling domain of the CAR of the invention triggersor elicits activation of at least one of the normal effector functionsof the immune cell in which the CAR has been placed. The term “effectorfunction” refers to a specialized function of a cell. The effectorfunction of a T cell may, for example, be cytolytic activity or helperactivity including the secretion of cytokines. While usually the entireintracellular signaling domain can be employed, in many cases it is notnecessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Examples of intracellular domain sequences that are of particular use inthe invention include those derived from an intracellular signalingdomain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fcreceptor subunit, an IL-2 receptor subunit, CD3ζ, FcRγ, FcRβ, CD3γ,CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRl, DAP10,and DAP12. It is particularly preferred that the intracellular signalingdomain in the CAR of the invention comprises a cytoplasmic signalingsequence derived from CD3ζ. Specifically, examples of the ITAM includeresidues 51 to 164 of CD3 (GenBank Accession No. NP_932170), residues 45to 86 of FcsRly (Gen Bank Accession No. NP_004097), residues 201 to 244of FcsRi (GenBank Accession No. NP_000130), residues 139 to 182 of CD3y(GenBank Accession No. NP_000064), residues 128 to 171 of CD35 (GenBankAccession No. NP_000723), residues 153 to 207 of CD3s (GenBank AccessionNo. NP_000724), residues 402 to 495 of CD5 (GenBank Accession No.NP_055022), residues 707 to 847 of CD22 (GenBank AccessionNo.NP_001762), residues 166 to 226 of CD79a (GenBank Accession No.NP_001774), residues 182 to 229 of CD79b (GenBank Accession No.NP_000611), and residues 177 to 252 of CD66d (GenBank Accession No.NP_001806), and their variants. The referenced residues are based onamino acid sequence information from GenBank and is based on the fulllength of the precursor (including a signal peptide sequence etc.) ofeach protein. Preferred examples of intracellular signaling domains foruse in the CAR of the invention include the cytoplasmic sequences of theT cell receptor (TCR) and co-receptors that act in concert to initiatesignal transduction following antigen receptor engagement, as well asany derivative or variant of these sequences and any synthetic sequencethat has the same functional capability.

In a preferred embodiment, the cytoplasmic domain of the CAR can bedesigned to comprise the CD3ζ signaling domain by itself or combinedwith any other desired cytoplasmic domain(s) useful in the context ofthe CAR of the invention. In one particular aspect, the CAR of thepresent invention comprises the CD3ζ signaling domain, preferablycomprising or consisting in the sequence of SEQ ID NO: 22 or a sequencehaving at least 80, 85, 90 or 95% of identity therewith.

The cytoplasmic domain of the CAR can comprise a CD3ζ chain portion anda costimulatory signaling region. The costimulatory signaling regionrefers to a portion of the CAR comprising the intracellular domain of acostimulatory molecule.

Examples of co-stimulatory molecules that may be used in the presentinvention include an MHC class I molecule, TNF receptor proteins,immunoglobulin-like proteins, cytokine receptors, integrin, signalinglymphocytic activation molecules (SLAM proteins), activating NK cellreceptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8),CD2, CD4, CDS, CD7, CD8a, CD8, CDl la, LFA-1 (CDl la/CD18), CDl lb, CDllc, CDl ld, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a,CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, CRTAM,OX40 (CD134), 4-1BB (CD137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55),SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS(CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4,ICAM-1, IL2Rβ, IL2Rγ, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM,ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C,NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PD-1, PSGLI, SLAMF6(NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA-I, VLA-6, aligand that specifically binds with CD83, and the like.

Stimulatory ligands are well-known in the art and encompass, inter alia,a MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, asuperagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

In one embodiment, the intracellular signaling domains comprise at leastone intracellular domain selected from an intracellular signaling domainof a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fcreceptor subunit, an IL-2 receptor subunit, CD3, FcRγ, FcRβ, CD3γ, CD3δ,CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRl, DAP10, and DAP12.

In one embodiment, the costimulatory domain of the CAR according to theinvention is selected from CD28, CD134 (TNFRSF4, OX40), CD154 (CD4OL),CD137 (4-1BB), ICOS (CD278), CD27, CD244 (2B4), CD149, DAP10, CD30,IL2-R, IL7r6, IL21-R, NKp30, NKp44, CD27 and DNAM-1. These differentco-stimulatory domains produce different cytokine profiles which, inturn, may produce effects on target cell-mediated cytotoxicity and thetumor microenvironment. Particularly, the costimulatory domain is fusedtogether with CD3ζ. In one embodiment, the costimulatory domain of theCAR according to the invention is selected from CD28, 4-1BB and OX40.

While any suitable endodomain can be used in the CAR of the invention,in certain embodiments, the invention specifically contemplates the useof all or a part of the endodomains of 4-1BB and CD3ζ as theintracellular domain. The cytoplasmic signaling sequences within theintracellular signaling domain of the CAR of the invention may be linkedto each other in a random or specified order. In a CAR containing morethan one intracellular endodomain, an oligopeptide linker, as describedabove, or a polypeptide linker can be inserted between the intracellularendodomains to link the domains. Optionally, a short oligo- orpolypeptide linker, preferably between 2 and 10 amino acids in lengthmay form the linkage. A glycine-serine doublet or continuous sequenceprovides a particularly suitable linker. Particularly, the peptidelinker may be a (Gly)₃-Ser linker or any repetition thereof such asdoublets or triplets thereof.

As used herein, the term “4-1BB costimulatory signaling region” refersto a specific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity,preferably 90% sequence identity, more preferably at least 95% sequenceidentity with the 4-1BB costimulatory signaling region sequence as shownin SEQ ID NO: 21. The example sequences of the 4-1BB costimulatorysignaling region are provided in U.S. 20130266551A1.

Examples of sequences of CD28 costimulatory signaling domain areprovided in U.S. Pat. No. 5,686,281; Geiger, T. L. et al., Blood 98:2364-2371 (2001); Hombach A. et al., J Immunol 167: 6123-6131 (2001);Maher J. et al. Nat Biotechnol 20: 70-75 (2002); Haynes N. M. et al., JImmunol 169: 5780-5786 (2002); Haynes N. M. et al., Blood 100: 3155-3163(2002).

Non-limiting example sequences of the OX40 costimulatory signalingregion are disclosed in U.S. 2012/20148552 A1.

In a particular embodiment, the CAR of the invention comprises a 4-1BBcostimulatory signaling region, preferably comprising or consisting inthe sequence of SEQ ID NO: 21 or a sequence having at least 80, 85, 90or 95% of identity therewith.

Signal Peptide

In addition to the antigen targeting domain, hinge domain, transmembranedomain, and signaling endodomain, the CAR of the invention can furthercomprise a signal peptide sequence linked to the N-terminus of the CAR.Signal peptide sequences exist at the N-terminus of many secretoryproteins and membrane proteins and have typically a length of 15 to 30amino acids. Since many of the protein molecules mentioned above havesignal peptide sequences, these signal peptides can be used as a signalpeptide for the CAR of this invention.

In an embodiment, the sequence comprising the antigen binding domainfurther comprises a signal peptide. In embodiments where the antigenbinding domain comprises an antigen binding fragment, preferably a scFv,the signal peptide may be positioned at the amino terminus of theantigen binding fragment or scFv. In some embodiments, when the heavychain variable region is N-terminal, the signal peptide may bepositioned at the amino terminus of the heavy chain variable region. Insome embodiments, when the light chain variable region is N-terminal,the leader sequence may be positioned at the amino terminus of the lightchain variable region. The leader sequence may comprise any suitablesignal sequence. In one embodiment, the CAR of the invention, preferablythe antibody or the antigen binding fragment, even more preferably thescFv, comprises a signal peptide selected from the group consisting ofCD8a, a mouse Ig Kappa signal peptide, a human IgG4 signal peptide, anIL2 signal peptide, a human IgG2 signal peptide and a Gaussia luc signalpeptide. In one embodiment, the CAR of the invention, preferably thescFv, comprises a signal peptide selected from the group consisting ofSEQ ID No: 17, 24, 75, 76, 77, and 78.

In a particular embodiment, the CAR of the invention comprises a CD8asignal peptide, preferably comprising or consisting in the sequence ofSEQ ID NO: 17 or a sequence having at least 80, 85, 90 or 95% ofidentity therewith.

Cleavable Linker

In a particular embodiment, the CAR according to the invention furthercomprises a cleavable linker. The cleavable linker may be a peptide, apolypeptide or a part of a polypeptide, which is cleaved after thegeneration of the protein or polypeptide, particularly, after thetranslation of the CAR according to the invention.

Particularly, the cleavable linker is a self-cleavable, self-cleaving,self-cleavage peptide or linker, these terms being used interchangeablyherein.

In one embodiment, the cleavable linker comprises a 2A peptide. “2A” or“2A-like” sequences are part of a large family of peptides that cancause peptide bond-skipping. Particularly, the mechanism of 2A-mediated“self-cleavage” was recently discovered to be ribosome skipping theformation of a glycyl-prolyl peptide bond at the C-terminus of the 2Apeptide. The 2A-peptide-mediated cleavage commences after thetranslation. Successful skipping and recommencement of translationresults in two “cleaved” proteins: the protein upstream of the 2A isattached to the complete 2A peptide except for the C-terminal proline,and the protein downstream of the 2A is attached to one proline at theN-terminus. Successful skipping but ribosome fall-off and discontinuedtranslation results in only the protein upstream of 2A. Several 2Apeptides have been identified in picornaviruses, insect viruses and typeC rotaviruses.

Examples of cleavable linker according to the invention include, but arenot limited to, porcine teschovirus-1 2A (P2A), FMDV 2A (F2A); equinerhinitis A virus (ERAV) 2A (E2A); and Thosea asigna virus 2A (T2A),cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flacherie Virus 2A(BmIFV2A), or a combination thereof, for example such as described inKim et al. (2011) PLoS ONE 6(4): e18556 and in Liu et al (2017) Sci Rep.2017; 7: 2193.

Preferably, the cleavable linker is P2A which comprises or consists ofthe sequence set forth in SEQ ID NO: 28 or a sequence having at least80, 85, 90 or 95% of identity therewith.

In one embodiment, the N-terminus of the cleavable linker is operablylinked to the C-terminus of the CAR endodomain and/or the C-terminus ofthe cleavable linker is operably linked to the N-terminus of a reporter.

Reporter

For safety reasons, for tracking purposes or for elimination of unwantedmodified CAR expressing cells, the CAR construct according to theinvention may further comprise a selectable marker or reporter.Particularly, CAR cells can be engineered with self-cleaving linker toco-express CARs with a reporter, to provide the possibility of selectingor eliminating the CAR cells for example by antibodies.

In a particular embodiment, the N terminus of the reporter is connectedto the anti-HLA-G CAR by a cleavable linker, such as a 2A linker asdescribed hereabove, to allow release of the reporter molecule. Thereporter can then be used to confirm successful delivery or expressionof the CAR construct to cells of interest.

Particularly, the reporter is selected in the group consisting of ac-myc tag, CD20, CD52 (Campath), truncated EGFR (EGFRt), truncated CD19(CD19t), or any part or combination thereof, or any other markermolecule that can be expressed and/or detected in a cell.

In a preferred embodiment, the reporter is a CD19 reporter, preferably ahuman truncated CD19 (hCD19t) reporter in which amino-acid from position314-556 have been removed (i.e 313 amino-acid removed). In a particularembodiment, the truncated human CD19 is the one synthesized by GeneArtcontaining the extracellular and transmembrane portions of human CD19(amino acid 1-313) known under the accession number NP_001171569.1.

Preferably, the reporter is a human truncated CD19 reporter whichcomprises or consists of the sequence set forth in SEQ ID NO: 28 or asequence having at least 80, 85, 90 or 95% of identity therewith.

Particular CARs of the Present Invention

In a particular embodiment, the CAR of the present invention comprises asignal peptide, preferably a CD8a signal peptide; an antigen bindingdomain specific of one or several HLA-G isoform(s), in particular as ascFv, preferably as detailed above; optionally a hinge, preferablycomprising or consisting of (i) a human IgG4 hinge domain, (ii) a humanIgG4 hinge domain and a CH3 human IgG4 domain or (iii) a mutated CH2human IgG4 domain, a human IgG4 hinge domain and a CH3 human IgG4 hingedomain or (iv) a CD28 hinge or (v) a CD8a hinge; a transmembrane domain,preferably a CD28 transmembrane domain; a costimulatory signalingregion, preferably selected from CD28, 4-1BB and OX40 costimulatorysignaling region, more preferably a 4-1BB costimulatory signalingregion; and a CD3ζ signaling domain, and optionally a cleavable linker,preferably a 2A linker, and a reporter, preferably a hCD19t reporter.

In one embodiment the CAR construct is a bispecific CAR construct thatcomprises (i) a domain comprising CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3)comprising a sequence of SEQ ID NO: 11, 12 and 13, respectively, (ii) adomain comprising CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) comprising asequence of SEQ ID NO: 14, 15 and 16, respectively, (iii) a domaincomprising CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) comprising a sequence ofSEQ ID NO: 5, 6 and 7, respectively, and (iv) a domain comprising CDR 1,2 and 3 (LCDR1, LCDR2, LCDR3) comprising a sequence of SEQ ID NO: 8, 9and 10, respectively, optionally wherein each CDR may optionallycomprise 1, 2, 3 or 4 amino acid substitutions, deletions or insertions.

More specifically, the CAR of the present invention may comprise orconsist in the polypeptide sequence of SEQ ID NO: 32, 33, 34, 35, 36,37, 83 or 85 or a sequence having at least 80, 85, 90 or 95% of identitywith the sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37, 83 or 85 andhaving the same functions.

CAR Variations

Included in the scope of the invention are functional portions of theinventive CARs described herein. The term “functional portion” when usedin reference to a CAR refers to any part or fragment of the CAR of theinvention, which part or fragment retains the biological activity of theCAR of which it is a part (the parent CAR). Functional portionsencompass, for example, those parts of a CAR that retain the ability torecognize antigen or target cells, or detect, treat, or prevent adisease, to a similar extent, the same extent, or to a higher extent, asthe parent CAR. In reference to the parent CAR, the functional portioncan comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%,95%, or more, of the parent CAR.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent CAR.Desirably, the additional amino acids do not interfere with thebiological function, e.g., recognize target cells, detect cancer, treator prevent cancer, etc. More desirably, the additional amino acidsenhance the biological activity, as compared to the biological activityof the parent CAR.

Included in the scope of the invention are functional variants orbiological equivalent of the inventive CARs or antibody describedherein. A functional variant can, for example, comprise the amino acidsequence of the parent polypeptide with at least one conservative aminoacid substitution. Alternatively or additionally, the functionalvariants can comprise the amino acid sequence of the parent polypeptidewith at least one non-conservative amino acid substitution. In thiscase, it is preferable for the non-conservative amino acid substitutionto not interfere with or inhibit the biological activity of thefunctional variant. The non-conservative amino acid substitution mayenhance the biological activity of the functional variant, such that thebiological activity of the functional variant is increased as comparedto the parent polypeptide.

Such biological variant (including functional portions thereof) cancomprise synthetic amino acids in place of one or morenaturally-occurring amino acids.

Such biological variant (including functional portions thereof) can beglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into anacid addition salt and/or optionally dimerized or polymerized, orconjugated.

Such biological variant (including functional portions thereof) can beobtained by methods known in the art. The polypeptides may be made byany suitable method of making polypeptides or proteins. Suitable methodsof de novo synthesizing polypeptides and proteins are described inreferences, such as Chan et al., Fmoc Solid Phase Peptide Synthesis,Oxford University Press, Oxford, United Kingdom, 2000; Peptide andProtein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; EpitopeMapping, ed. Westwood et al., Oxford University Press, Oxford, UnitedKingdom, 2001 and U.S. Pat. No. 5,449,752. Also, polypeptides andproteins can be recombinantly produced using the nucleic acids describedherein using standard recombinant methods. See, for instance, Sambrooket al, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, NY, 1994.

Nucleic Acid Construct and Vectors for CAR Expression

The invention also relates to a nucleic acid encoding an antibody asdescribed above or a nucleic acid encoding a CAR according to theinvention. Such nucleic acid can be transduced into a cell, inparticular an immune cell, to create a cell that expresses the CAR.Particularly, the nucleic acid construct comprises sequences encoding anexternal domain, an intracellular domain, a transmembrane, optionally ahinge domain, and optionally a cleavable linker, a reporter and/or asignal peptide as described hereabove.

In one embodiment, the nucleic acid construct sequentially comprises orconsists in, from N to C terminus: optionally a peptide signal sequence,an anti-HLA-G antibody or fragment thereof, preferably an anti-HLA-GscFv, a spacer domain, a transmembrane domain, at least oneintracellular domain, and optionally a cleavable linker and a reporter.

In a yet further embodiment, the nucleic acid construct furthercomprises a tag, preferably a Flag tag. For example, the Flag tagcomprises or consists in SEQ ID NO: 23, SEQ ID NO: 60, or SEQ ID NO: 79.

In some embodiments, the nucleic acid construct comprises:

(i) a nucleic acid sequence encoding an anti-HLA-G scFv as describedabove;

(ii) optionally a nucleic acid sequence encoding a hinge, preferablyselected from the group consisting of (i) CD28 hinge, (ii) CD8 alphahinge, (iii) a human IgG4 hinge domain, (iv) a human IgG4 hinge domainand a CH3 human IgG4 domain or (v) a mutated CH2 human IgG4 domain, ahuman IgG4 hinge domain and a CH3 human IgG4 hinge domain

(iii) a nucleic acid sequence encoding a transmembrane domain,preferably a CD28 transmembrane domain;

(iii) a nucleic acid sequence encoding an endodomain, preferably a 4-1BBdomain and/or a CD3ζ domain;

(iv) optionally a cleavable linker, preferably a P2A cleavable linker;

(v) optionally a reporter, preferably a hCD19t reporter; and/or

(vi) optionally a signal peptide, preferably selected from the groupconsisting of CD8a, a mouse Ig Kappa signal peptide, a human IgG4 signalpeptide and an IL2 signal peptide.

In some embodiments, the nucleic acid construct comprises:

(i) a nucleic acid sequence encoding the HCDR1, HCDR2 and HCDR 3 of SEQID NO: 5, 6 and 7, respectively, for instance SEQ ID NO: 42, 43 and 44,respectively; and

(ii) a nucleic acid sequence encoding the LCDR1, LCDR2 and LCDR3 of SEQID NO: 8, 9 and 10, respectively, for instance SEQ ID NO: 45, 46 and 47respectively.

Alternatively, the nucleic acid construct comprises:

(i) a nucleic acid sequence encoding the HCDR1, HCDR2 and HCDR 3 of SEQID NO: 11, 12 and 13, respectively, for instance SEQ ID NO: 48, 49 and50, respectively, and

(ii) a nucleic acid sequence encoding the LCDR1, LCDR2 and LCDR3 of SEQID NO: 14, 15 and 16, respectively, for instance SEQ ID NO: 51, 52 and53, respectively.

In a particular embodiment, the nucleic acid construct comprises oressentially consists in:

(i) a nucleic acid sequence encoding SEQ ID NO: 1 or a sequence havingat least 80, 85, 90 or 95% identity with SEQ ID NO: 1, for instance SEQID NO: 38;

(ii) a nucleic acid sequence encoding SEQ ID NO: 2 or a sequence havingat least 80, 85, 90 or 95% identity with SEQ ID NO: 2, for instance SEQID NO: 39;

(iii) optionally a nucleic acid sequence encoding (a) SEQ ID NO: 18, (b)SEQ ID 19, (c) SEQ ID NO: 25, (d) SEQ ID NO: 25 and SEQ ID NO: 27, or(e) SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, or a sequence havingat least 80, 85, 90 or 95% identity thereto, for instance (a) SEQ ID NO:55, (b) SEQ ID NO: 56, (c) SEQ ID NO: 62, (d) SEQ ID NO: 62 and SEQ IDNO: 64, or (e) SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO:64,respectively;

(iv) a nucleic acid sequence encoding a transmembrane domain, preferablya CD28 transmembrane domain, even more preferably SEQ ID NO: 57 or SEQID NO:80;

(v) a nucleic acid sequence encoding an endodomain, preferably a 4-1BBdomain, for instance SEQ ID NO: 58 or 81, and/or a CD3ζ endodomain, forinstance SEQ ID NO: 59 or 82, preferably a 4-1BB domain and a CD3ζendodomain;

(vi) optionally a nucleic acid sequence encoding a cleavable linker,preferably a P2A cleavable linker, even more preferably encoding SEQ IDNO: 28 or a sequence having at least 80, 85, 90 or 95% identity with SEQID NO: 28, for instance SEQ ID NO: 65;

(vii) optionally a nucleic acid sequence encoding a reporter, preferablya hCD19t reporter, even more preferably encoding SEQ ID NO: 29 or asequence having at least 80, 85, 90 or 95% identity with SEQ ID NO: 29,for instance SEQ ID NO: 66;

(viii) optionally a nucleic acid sequence encoding a signal peptide,preferably a CD8 cc signal peptide, even more preferably SEQ ID NO. 54or 61.

In a yet further embodiment, the nucleic acid construct furthercomprises a nucleic acid sequence encoding a tag, preferably a Flag tag,for instance of SEQ ID NO: 23 or a sequence having at least 80, 85, 90or 95% identity thereto.

In another particular embodiment, the nucleic acid construct comprisesor essentially consists in:

(i) a nucleic acid sequence encoding SEQ ID NO: 3 or a sequence havingat least 80, 85, 90 or 95% identity with SEQ ID NO: 3; for instance, SEQID NO: 40; and/or

(ii) a nucleic acid sequence encoding SEQ ID NO: 4 or a sequence havingat least 80, 85, 90 or 95% identity with SEQ ID NO: 4; for instance, SEQID NO: 41;

(iii) optionally a nucleic acid sequence encoding (a) SEQ ID NO: 18, (b)SEQ ID 19, (c) SEQ ID NO: 25, (d) SEQ ID NO: 25 and SEQ ID NO: 27, or(e) SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, or a sequence havingat least 80, 85, 90 or 95% identity thereto, for instance (a) SEQ ID NO:55, (b) SEQ ID NO: 56, (c) SEQ ID NO: 62, (d) SEQ ID NO: 62 and SEQ IDNO: 64, or (e) SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID N0:64,respectively;

(iv) a nucleic acid sequence encoding a transmembrane domain, preferablya CD28 transmembrane domain, even more preferably SEQ ID NO: 57 or SEQID NO:80;

(v) a nucleic acid sequence encoding an endodomain, preferably a 4-1BBdomain, for instance SEQ ID NO: 58 or 81, and/or a CD3ζ endodomain, forinstance SEQ ID NO: 59 or 82, preferably a 4-1BB domain and a CD3ζendodomain;

(vi) optionally a nucleic acid sequence encoding a cleavable linker,preferably a P2A cleavable linker, even more preferably encoding SEQ IDNO: 28 or a sequence having at least 80, 85, 90 or 95% identity with SEQID NO: 28, for instance SEQ ID NO: 65;

(vii) optionally a nucleic acid sequence encoding a reporter, preferablya hCD19t reporter, even more preferably encoding SEQ ID NO: 29 or asequence having at least 80, 85, 90 or 95% identity with SEQ ID NO: 29,for instance SEQ ID NO: 66;

(viii) optionally a nucleic acid sequence encoding a signal peptide,preferably a CD8 a, signal peptide, even more preferably SEQ ID NO. 54or 61.

In a yet further embodiment, the nucleic acid construct furthercomprises a nucleic acid sequence encoding a tag, preferably a Flag tag,for instance of SEQ ID NO: 23 or a sequence having at least 80, 85, 90or 95% identity thereto.

In another particular embodiment, the nucleic acid constructsequentially comprises or consists in from N to C terminus:

(i) optionally a signal peptide, preferably a CD8a signal peptide, evenmore preferably SEQ ID NO: 54 or 61,

(ii) a nucleic acid sequence encoding SEQ ID NO: 30 or a sequence havingat least 80, 85, 90 or 95% identity with SEQ ID NO: 30; for instance SEQID NO: 67; or a nucleic acid sequence encoding SEQ ID NO: 31 or asequence having at least 80, 85, 90 or 95% identity with SEQ ID NO: 31;for instance SEQ ID NO: 68;

(iii) optionally a nucleic acid sequence encoding (a) SEQ ID NO: 18, (b)SEQ ID 19, (c) SEQ ID NO: 25, (d) SEQ ID NO: 25 and SEQ ID NO: 27, or(e) SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, or a sequence havingat least 80, 85, 90 or 95% identity thereto, for instance (a) SEQ ID NO:55, (b) SEQ ID NO: 56, (c) SEQ ID NO: 62, (d) SEQ ID NO: 62 and SEQ IDNO: 64, or (e) SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID N0:64,respectively;

(iv) a nucleic acid sequence encoding a transmembrane domain, preferablya CD28 transmembrane domain, even more preferably SEQ ID NO: 57 or SEQID NO:80;

(v) a nucleic acid sequence encoding an endodomain, preferably a 4-1BBdomain, for instance SEQ ID NO: 58 or 81, and/or a CD3ζ endodomain, forinstance SEQ ID NO: 59 or 82, preferably a 4-1BB domain and a CD3ζendodomain;

(vi) optionally a nucleic acid sequence encoding a cleavable linker,preferably a P2A cleavable linker, even more preferably encoding SEQ IDNO: 28 or a sequence having at least 80, 85, 90 or 95% identity with SEQID NO: 28, for instance SEQ ID NO: 65;

(vii) optionally a nucleic acid sequence encoding a reporter, preferablya hCD19t reporter, even more preferably encoding SEQ ID NO: 29 or asequence having at least 80, 85, 90 or 95% identity with SEQ ID NO: 29,for instance SEQ ID NO: 66.

In a particular embodiment, the present invention provides a nucleicacid sequence encoding a particular CAR of the present invention,preferably a CAR describe in the sequence set forth in SEQ ID No: 32,33, 34, 35, 36, 37, 83 or 85, more preferably such a nucleic acidsequence or construct comprising or consisting of SEQ ID NO: 69, 70, 71,72, 73, 74, 84 or 86 or a sequence having at least 80, 85, 90 or 95%identity thereto.

The sequence of the open reading frame encoding the chimeric receptorcan be obtained from a genomic DNA source, a cDNA source, generated byPCR from a cDNA source or else. Otherwise it can be chemicallysynthesized or combinations thereof. Depending upon the size of thegenomic DNA and the number of introns, it may be desirable to use cDNAor a combination thereof as it is found that introns stabilize the mRNAor provide immune cell, particularly T cell-specific expression (Bartheland Goldfeld, 2003). Also, it may be further advantageous to useendogenous or exogenous non-coding regions to stabilize the mRNA. Forexpression of a chimeric antigen receptor of the present invention, thenaturally occurring or endogenous transcriptional initiation region ofthe nucleic acid sequence encoding N-terminal components of the chimericreceptor can be used to generate the chimeric receptor in the targethost cell. Alternatively, an exogenous transcriptional initiation regioncan be used that allows for constitutive or inducible expression,wherein expression can be controlled depending upon the target host, thelevel of expression desired, the nature of the target host, and thelike.

Likewise, a signal sequence directing the chimeric receptor to thesurface membrane can be the endogenous signal sequence of N-terminalcomponent of the chimeric receptor. Optionally, in some instances, itmay be desirable to exchange this sequence for a different signalsequence. However, the signal sequence selected should be compatiblewith the secretory pathway of the immune cell that will express the CARso that the chimeric receptor is presented on the surface of the cell.

In accordance with the present invention, the nucleic acid construct istransformed or introduced into a cell and is transcribed and translatedto produce a product (i.e. a chimeric receptor). Thus, the nucleic acidconstruct can further include at least one promoter for directingtranscription of the CAR.

In one embodiment, the promoter is operably linked to the nucleic acidsequence encoding the chimeric receptor of the present invention, i.e.,they are positioned so as to promote transcription of the messenger RNAfrom the DNA encoding the chimeric receptor. The promoter can be ofgenomic origin or synthetically generated. A variety of promoters foruse in immune cells and particularly in T cells are well-known in theart (e.g., the CD4 promoter disclosed by Marodon et al. (2003)). Thepromoter can be constitutive or inducible, where induction is associatedwith the specific cell type or a specific level of maturation, or drug(e.g., tetracycline or doxorubicin) for example. Examples of induciblepromoters include, but are not limited to, a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter. Alternatively, a number of well-known viral promoters are alsosuitable. Promoters of interest include the β-actin promoter, SV40 earlyand late promoters, immunoglobulin promoter, human cytomegaloviruspromoter, retrovirus promoter, and the Friend spleen focus-forming viruspromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avianleukemia virus promoter, Cytomegalovirus immediate early promoter, Roussarcoma virus promoter, as well as human gene promoters such as, but notlimited to, the actin promoter, the myosin promoter, the hemoglobinpromoter, and the creatine kinase promoter. The promoters may or may notbe associated with enhancers, wherein the enhancers may be naturallyassociated with the particular promoter or associated with a differentpromoter.

Similarly, a termination region may be provided by the naturallyoccurring or endogenous transcriptional termination region of thenucleic acid sequence encoding the C-terminal component of the chimericreceptor. Alternatively, the termination region may be derived from adifferent source. For the most part, the source of the terminationregion is generally not considered to be critical to the expression of arecombinant protein and a wide variety of termination regions can beemployed without adversely affecting expression. As will be appreciatedby one skilled in the art that, in some instances, a few amino acids atthe ends of the antigen binding domain in the CAR can be deleted,usually not more than 10, more usually not more than 5 residues, forexample. Also, it may be desirable to introduce a small number of aminoacids at the borders, usually not more than 10, more usually not morethan 5 residues. The deletion or insertion of amino acids may be as aresult of the needs of the construction, providing for convenientrestriction sites, ease of manipulation, improvement in levels ofexpression, or the like. In addition, the substitute of one or moreamino acids with a different amino acid can occur for similar reasons,usually not substituting more than about five amino acids in any onedomain.

In another embodiment, the nucleic acid construct further comprises apromoter, the correct translation initiation sequence such as aribosomal binding site and a start codon, a termination codon, and atranscription termination sequence.

The nucleic acid construct according to the invention may also compriseother regulatory regions such as enhancers, silencers and boundaryelements/insulators to direct the level of transcription of a givengene. The nucleic acid construct that encodes the chimeric receptoraccording to the invention can be prepared in conventional ways.Because, for the most part, natural sequences may be employed, thenatural genes may be isolated and manipulated, as appropriate, so as toallow for the proper joining of the various components. Thus, thenucleic acid sequences encoding for the N-terminal and C-terminalproteins of the chimeric receptor can be isolated by employing thepolymerase chain reaction (PCR), using appropriate primers that resultin deletion of the undesired portions of the gene. Alternatively,restriction digests of cloned genes can be used to generate the chimericconstruct. In either case, the sequences can be selected to provide forrestriction sites that are blunt-ended, or have complementary overlaps.

The various manipulations for preparing the chimeric construct can becarried out in vitro and in particular embodiments the chimericconstruct is introduced into vectors for cloning and expression in anappropriate host using standard transformation or transfection methods.Thus, after each manipulation, the resulting construct from joining ofthe DNA sequences is cloned, the vector isolated, and the sequencescreened to ensure that the sequence encodes the desired chimericreceptor. The sequence can be screened by restriction analysis,sequencing, or the like.

CARs may be prepared using expression vectors. Aspects of the presentdisclosure relate to a nucleic acid sequence encoding a HLA-G CAR andvectors comprising a nucleic acid sequence encoding the CAR as describedabove.

For example, the nucleic acid construct according to the invention canbe cloned into a vector including, but not limited to, a plasmid, aphagemid, a phage derivative, a virus, and a cosmid. Vectors ofparticular interest include expression vectors, replication vectors,probe generation vectors, and sequencing vectors. According to thenucleic acid construct and the host cell, the vector according to theinvention can comprise: a promoter, a terminator, replication origin,selectable markers, multiple cloning sites, packaging sites and thelike. In some other embodiments, the vector comprises, or alternativelyconsists essentially thereof, or yet further consists of, a Kozakconsensus sequence upstream of the sequence encoding the CAR. In someembodiments, the vector comprises a polynucleotide conferring antibioticresistance.

Particularly, the vector according to the invention may be provided to acell in the form of a viral vector. A number of viral based systems havebeen developed for gene transfer into mammalian cells. Viral vectortechnology is well known in the art and is described, for example, inSambrook, et al. ((2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in other virology and molecularbiology manuals. Viruses, which are useful as vectors include, but arenot limited to, retroviruses, adenoviruses, adeno-associated viruses,herpesviruses and lentiviruses. Preferably, the vector is an IntegrativeLentiviral Vector. Lentiviral vectors have the added advantage overvectors derived from oncoretroviruses in that they can transducenon-proliferating cells and present low immunogenicity. In general, asuitable vector contains an origin of replication functional in at leastone organism, a promoter sequence, convenient restriction endonucleasesites, and one or more selectable markers such as described in WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193.

Particularly, in order to assess the expression of a CAR, the expressionvector to be introduced into a cell can also contain either a selectablemarker gene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors, in other aspects, theselectable marker may be carried on a separate piece of DNA and used ina co-transfection procedure. Both selectable markers and reporter genesmay be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers include, forexample, antibiotic-resistance genes and the like.

In one aspect, the term “vector” intends a recombinant vector thatretains the ability to infect and transduce non-dividing and/orslowly-dividing cells and integrate into the target cell's genome. Inseveral aspects, the vector is derived from or based on a wild-typevirus. In further aspects, the vector is derived from or based on awild-type retrovirus. Particularly, the retrovirus can be selected froma leukemia virus such as a Moloney Murine Leukemia Virus (MMLV), theHuman Immunodeficiency Virus (HIV), or the Gibbon Ape Leukemia virus(GALV). The foreign enhancer and promoter may be the humancytomegalovirus (HCMV) immediate early (IE) enhancer and promoter, theenhancer and promoter (U3 region) of the Moloney Murine Sarcoma Virus(MMSV), the U3 region of Rous Sarcoma Virus (RSV), the U3 region ofSpleen Focus Forming Virus (SFFV), or the HCMV IE enhancer joined to thenative Moloney Murine Leukemia Virus (MMLV) promoter. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Viral vectors canalso be derived from lentivirus, poxviruses, herpes simplex virus I(HSV), Epstein Barr virus (EBV), papillomavirus, adenoviruses andadeno-associated viruses, and the like. See for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

Preferably, the vector according to the invention is a lentiviralvector. Particularly, the vector is derived from primate and non-primatelentivirus. Examples of primate lentiviruses include the humanimmunodeficiency virus (HIV), the causative agent of human acquiredimmunodeficiency syndrome (AIDS), and the simian immunodeficiency virus(SIV). The non-primate lentiviral group includes the prototype “slowvirus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV). Prior art recombinantlentiviral vectors are known in the art, e.g., see U.S. Pat. Nos.6,924,123; 7,056,699; 7,07,993; 7,419,829 and 7,442,551, incorporatedherein by reference.

Commercial retroviral vectors for use in this disclosure include, butare not limited to, pFB-neo vectors (STRATAGENE®), Invitrogen's pLentiseries versions 4, 6, and 6.2 “ViraPower” system. Manufactured byLentigen Corp.; pHIV-7-GFP, lab generated and used by the City of HopeResearch Institute; “Lenti-X” lentiviral vector, pLVX, manufactured byClontech; pLKO.1-puro, manufactured by Sigma-Aldrich; pLemiR,manufactured by Open Biosystems; and pLV, lab generated and used by theCharité Medical School, Institute of Virology (CBF), Berlin, Germany.

It will be evident that a viral vector according to the disclosure neednot be confined to the components of a particular virus. The viralvector may comprise components derived from two or more differentviruses, and may also comprise synthetic components. Vector componentscan be manipulated to obtain desired characteristics, such as targetcell specificity.

U.S. Pat. No. 6,924,123 discloses that certain retroviral sequencefacilitate integration into the target cell genome. This patent teachesthat each retroviral genome comprises genes called gag, pol and envwhich code for virion proteins and enzymes. These genes are flanked atboth ends by regions called long terminal repeats (LTRs). The LTRs areresponsible for proviral integration, and transcription. They also serveas enhancer-promoter sequences. Accordingly, the vector according to theinvention can comprise one or more of the integration features.

In some embodiment, the components of the particles not encoded by thevector genome are provided in trans by additional nucleic acid sequences(the “packaging system”, which usually includes either or both of thegag/pol and env genes) expressed in the host cell, for example using ahelper virus strategy. The set of sequences required for the productionof the viral vector particles may be introduced into the host cell bytransient transfection, or they may be integrated into the host cellgenome, or they may be provided in a mixture of ways, such as helpersequences. The techniques involved are known to those skilled in theart. For example, the retroviral constructs are packaging plasmidscomprising at least one retroviral helper DNA sequence derived from areplication-incompetent retroviral genome encoding in trans all virionproteins required to package a replication incompetent retroviralvector, and for producing virion proteins capable of packaging thereplication-incompetent retroviral vector at high titer, without theproduction of replication-competent helper virus.

In the packaging process, the packaging plasmids and retroviral vectorsexpressing the CAR according to the invention are transientlyco-transfected into a first population of mammalian cells that arecapable of producing virus, such as human embryonic kidney cells, forexample 293 cells (ATCC No. CRL1573, ATCC, Rockville, Md.) to producehigh titer recombinant retrovirus-containing supernatants. In anothermethod of the invention this transiently transfected first population ofcells is then co-cultivated with mammalian target cells, for examplehuman lymphocytes, to transduce the target cells with the foreign geneat high efficiencies. In yet another method of the invention thesupernatants from the above described transiently transfected firstpopulation of cells are incubated with mammalian target cells, forexample human lymphocytes or hematopoietic stem cells, to transduce thetarget cells with the foreign gene at high efficiencies.

The nucleic acid construct according to the invention be inserted into avector and packaged in retroviral or lentiviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to cells of the subject either in vivo or ex vivo. In aparticular aspect, the packaging plasmids are stably expressed in afirst population of mammalian cells that are capable of producing virus,such as human embryonic kidney cells, for example 293 cells. Retroviralor lentiviral vectors are introduced into cells by eitherco-transfection with a selectable marker or infection with pseudotypedvirus. In both cases, the vectors integrate. Alternatively, vectors canbe introduced in an episomally maintained plasmid.

Anti-HLA-G CAR Expressing Cells

The present invention relates to a cell expressing a CAR as describedherein. The cell may be of any kind, including an immune cell capable ofexpressing the CAR or a cell, such as a bacterial cell, that harbors anexpression vector that encodes the CAR.

In the context of expressing a heterologous nucleic acid sequenceencoding the CAR of the invention, “host cell” refers to a cell that iscapable of replicating a vector and/or expressing a heterologous geneencoded by a vector. The term “cell” also includes their progeny, whichis any and all subsequent generations. It is understood that all progenymay not be identical due to deliberate or inadvertent mutations. A hostcell may be transfected by the nucleic acid construct or vectoraccording to the invention. A transformed cell includes the primarysubject cell and its progeny. As used herein, the terms “engineered” and“recombinant” cells or host cells are intended to refer to a cell intowhich an exogenous nucleic acid sequence, such as, for example, avector, more particularly a CAR, has been introduced. Therefore,recombinant cells are distinguishable from naturally occurring cellswhich do not contain a recombinantly introduced nucleic acid.

Upon transduction of a cell with a nucleic acid construct encoding aCAR, the cell will recognize the HLA-G isoform specified by the CAR.Thus, the invention also relates to cells expressing the CARs accordingto the invention. Particularly, the anti-HLA-G CAR expressing cellsaccording to the invention are immune cells able to express a CARaccording to the invention. In certain embodiments, immune cells aretransfected to comprise at least a CAR of the present invention.

The cell can comprise a CAR that specifically binds to β2M-associatedHLA-G isoforms, preferably to both HLA-G1 and HLA-G5 isoforms.Alternatively, the cell may comprise a CAR that specifically binds toβ2M-free HLA-G isoforms, preferably to both HLA-G2 and HLA-G6 isoformsand/or HLA-G1/β2M free and/or HLA-G5/β2M free isoforms.

In one embodiment, the cell expresses at least two different CARs. Forinstance, the cell may comprise a CAR that specifically binds toβ2M-associated HLA-G isoforms preferably to both HLA-G1 and HLA-G5, andanother CAR that specifically binds to a different antigen. The cell mayalternatively comprise a CAR that specifically binds to β2M-free HLA-Gisoforms, preferably to HLA-G2 and HLA-G6, and/or HLA-G1/β2M free and/orHLA-G5/β2M free isoforms and another CAR that specifically binds to adifferent antigen. In particular, the cell may comprise both a CAR thatspecifically binds to β2M-associated HLA-G isoforms preferably to bothHLA-G1 and HLA-G5, and a CAR that specifically binds to β2M-free HLA-Gisoforms, preferably to HLA-G2 and HLA-G6, and/or HLA-G1/β2M free and/orHLA-G5/β2M free isoforms.

Preferably, the cell comprises a CAR comprising an antigen bindingfragment derived from the LFTT-1 antibody as described hereabove and aCAR comprising an antigen binding fragment derived from the 15E7antibody as described hereabove.

Preferably such cell thus comprises a first CAR comprising (i) one heavychain comprising CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) comprising asequence of SEQ ID NO: 11, 12 and 13, respectively, and (ii) one lightchain comprising CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) comprising asequence of SEQ ID NO: 14, 15 and 16, respectively, and a second CARthat comprises (i) one heavy chain comprising CDR 1, 2 and 3 (HCDR1,HCDR2, HCDR3) comprising a sequence of SEQ ID NO: 5, 6 and 7,respectively, and (ii) one light chain comprising CDR 1, 2 and 3 (LCDR1,LCDR2, LCDR3) comprising a sequence of SEQ ID NO: 8, 9 and 10,respectively, optionally wherein each CDR may optionally comprise 1, 2,3 or 4 amino acid substitutions, deletions or insertions. Even morepreferably, such cell thus comprises a first CAR comprising orconsisting of the sequence set forth in SEQ ID NO: 32, 33, 34 or 85 or asequence having at least 80, 85, 90 or 95% identity therewith and asecond CAR comprising or consisting of SEQ ID NO: 35, 36, 37 or 83 or asequence having at least 80, 85, 90 or 95% identity therewith.

Alternatively, the cell can express a bispecific CAR comprising abispecific antigen binding domain as described hereabove that comprisesa domain that recognizes HLA-G/β2M free isoforms, preferably both HLA-G2and HLA-G6 and/or both HLA-G1/β2M free and HLA-G5/β2M free isoforms, anda second domain that recognizes HLA-G isoforms associated with the β2Mdomain preferably both HLA-G1 and HLA-G5.

Optionally, the cell expressing a CAR anti-HLA-G according to theinvention further expresses a CAR that targets an antigen involved in adisease, preferably such as cancer or viral infection, preferably anantigen targeted in cancer therapies or in viral therapies. It will beunderstood that such antigen is not HLA-G.

The cell according to the invention can be a prokaryotic or a eukaryoticcell. Preferably, the cells are eukaryotic cells, such as mammaliancells, and typically are human, feline or canine cells, more typicallyhuman cells, preferably primary human cells.

Preferably, the cells are immune cells. The cells can be selected from agroup consisting of a T cell, including CD4⁺ T cell, and CD8⁺ T cell, Bcell, NK cell, NKT cell, monocyte and dendritic cell, preferably thecell being a T cell, a B cell and NK cell.

The cells can be autologous cells, syngeneic cells, allogeneic cells andeven in some cases, xenogeneic cells. For instance, suitable immunecells that can be used in the invention include autologous T lymphocytecells, allogeneic T cells, xenogeneic T cells, progenitors of any of theforegoing, transformed tumor or xenogeneic immunologic effector cells,tumor infiltrating lymphocytes (TILs), cytotoxic lymphocytes or othercells that are capable of killing target cells when activated.

The immune cells may be isolated from human or non-human subjects or maybe derived from stem cells, e.g., embryonic stem cells, multipotent stemcells, pluripotent stem cells, induced pluripotent stem cells (iPSCs),adult stem cells or other reprogrammed cells. The cells for introductionof the nucleic acid constructs described herein may be isolated from asample, such as a biological sample, e.g., one obtained from or derivedfrom a subject. In some embodiments, the subject from which the cell isisolated is one having the disease or condition or in need of a celltherapy or to which cell therapy will be administered. The subject insome embodiments is a human in need of a particular therapeuticintervention, such as the adoptive cell therapy for which cells arebeing isolated, processed, and/or engineered. In some embodiments, thecells are derived from the blood, bone marrow, lymph, or lymphoidorgans, are cells of the immune system, such as cells of the innate oradaptive immune systems, e.g., myeloid or lymphoid cells, includinglymphocytes, typically T cells and/or NK cells.

In some embodiments, the cells include one or more subsets of T cells orother cell types, such as whole T cell populations, CD4+ cells, CD8+cells, and subpopulations thereof, such as those defined by function,activation state, maturity, potential for differentiation, expansion,recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation.

In certain embodiments, the immune cell is a T cell, e.g., an animal Tcell, a mammalian T cell, a feline T cell, a canine T cell or a human Tcell. Among the sub-types and subpopulations of T cells and/or of CD4+and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF),memory T cells and sub-types thereof, such as stem cell memory T (TSCM),central memory T (TCM), effector memory T (TEM), or terminallydifferentiated effector memory T cells, tumor-infiltrating lymphocytes(TIL), immature T cells, mature T cells, helper T cells, cytotoxic Tcells, mucosa-associated invariant T (MAIT) cells, naturally occurringand adaptive regulatory T (Treg) cells, helper T cells, such as TH1cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, α/β T cells, and δ/γ T cells. Non-limitingexamples of commercially available T-cell lines include lines BCL2 (AAA)Jurkat (ATCC® CRL-2902™), BCL2 (S70A) Jurkat (ATCC® CRL-2900™), BCL2(S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat (ATCC® CRL-2899™), NeoJurkat (ATCC® CRL-2898™) TALL-104 cytotoxic human T cell line (ATCC#CRL-11386). Further examples include but are not limited to matureT-cell lines, e.g., such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102,Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1 and T34; and immatureT-cell lines, e.g., ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528,EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37,KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13,MOLT-16, MT-1, MT-ALL, P12/Ichikawa, Peer, PER0117, PER-255, PF-382,PFI-285, RPMI-8402, ST-4, SUP-T1 to T14, TALL-1, TALL-101, TALL-103/2,TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3,and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCCCRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4; 11 (ATCC CRL-1873), CCRF-CEM(ATCC CRM-CCL-119); and cutaneous T-cell lymphoma lines, e.g., HuT78(ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCCTIB-162).Non-limiting exemplary sources for such commercially available celllines include the American Type Culture Collection, or ATCC,(http://www.atcc.org/) and the German Collection of Microorganisms andCell Cultures (https://www.dsmz.de/).

In some embodiments, the cells are natural killer (NK) cells, NaturalKiller T (NKT) cells, cytokine-induced killer (CIK) cells,tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer (LAK)cells, or the like. NK cells may either be isolated or obtained from acommercially available source. Non-limiting examples of commercial NKcell lines include lines NK-92 (ATCC® CRL-2407™), NK-92MI (ATCC®CRL-2408™). Further examples include but are not limited to NK linesHANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplarysources for such commercially available cell lines include the AmericanType Culture Collection, or ATCC, (http://www.atcc.org/) and the GermanCollection of Microorganisms and Cell Cultures (https://www.dsmz.de/).

In a particular embodiment, the host cell presenting the CAR accordingto the invention is selected from cytotoxic T cells (also known as TC,Cytotoxic T Lymphocyte, CTL, T Killer cell, a lytic T cell, CD8+ T cellsor killer T cell) and NK cells.

In some embodiments, the cells are B cells, monocytes or granulocytes,e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mastcells, eosinophils, and/or basophils.

Method for Preparing CAR Expressing Cells

Methods of introducing genes into a cell and expressing genes in a cellare known in the art.

Particularly, methods for producing cells comprising vectors and/orexogenous nucleic acids are well-known in the art. See, for example,Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York). In the context of an expressionvector, the vector can be readily introduced into a host cell, e.g.,mammalian, bacterial, yeast, or insect cell by any method in the art.For example, the expression vector can be transferred into a host cellby physical, chemical, or biological means that are more particularlydescribed here below.

In some embodiments, the methods include isolating cells from thesubject, preparing, processing, culturing, and/or engineering them, asdescribed herein, and re-introducing them into the same patient, beforeor after cryopreservation.

Here is particularly provided a method of producing anti-HLA-G CARexpressing cells comprising, or alternatively consisting essentially of,or yet further consisting of the steps: (i) transducing a population ofisolated cells with a nucleic acid sequence encoding the CAR asdescribed herein; and (ii) selecting a subpopulation of said isolatedcells that have been successfully transduced with said nucleic acidsequence of step (i) thereby producing anti-HLA-G CAR expressing cells.In one aspect, the isolated cells are selected from a group consistingof T cells and NK cells.

Here is even more particularly provided a method of producing anti-HLA-GCAR expressing cells comprising, or alternatively consisting essentiallyof, or yet further consisting of the steps: (i) acquisition of an immunecell population (e.g. blood cells) (ii) isolation of a particular cellpopulation (e.g. T cells and/or NK cells) (iii) transducing a populationof isolated cells with a nucleic acid sequence encoding the CAR asdescribed herein; and (ii) selecting a subpopulation of said isolatedcells that have been successfully transduced with said nucleic acidsequence of step (iii) thereby producing anti HLA-G CAR expressingcells. These different steps are more particularly described below.

Cell Acquisition

Prior to expansion and genetic modification of the cells disclosedherein, cells may be obtained from a subject—for instance, inembodiments involving autologous therapy—or from a commerciallyavailable culture.

The cell can be acquired from samples include tissue, body fluid (e.g.blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat), and other samples taken directly from the subject, as well assamples resulting from one or more processing steps, such as separation,centrifugation, genetic engineering (e.g. transduction with viralvector), washing, and/or incubation.

Cells can be obtained from a number of non-limiting sources includingwhole blood, peripheral blood mononuclear cells (PBMCs), leukocytes,bone marrow, thymus tissue, lymph node tissue, cord blood, tissue from asite of infection, ascites, pleural effusion, tissue biopsy, tumor,leukemia, lymphoma, gut associated lymphoid tissue, mucosa associatedlymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach,intestine, colon, kidney, pancreas, breast, bone, prostate, cervix,testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.Samples include, in the context of cell therapy, e.g. adoptive celltherapy, samples from autologous and allogeneic sources.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contain cells other thanred blood cells and platelets.

In some embodiments, any number of T cell ok NK cell lines available andknown to those skilled in the art, such as described hereabove may beused. In some embodiments, cells can be derived from a healthy donor,from a patient diagnosed with cancer, from a patient diagnosed with anautoimmune or inflammatory disorder or from a patient diagnosed with aninfection. In some embodiments, cells can be part of a mixed populationof cells which present different phenotypic characteristics.

Cell Isolation

As is known to one of skill in the art, various methods are readilyavailable for isolating immune cells from a subject or can be adapted tothe present application, for example using Life Technologies Dynabeads®system; STEMcell Technologies EasySep™, RoboSep™, RosetteSep™, SepMate™;Miltenyi Biotec MACS™ cell separation kits, cell surface markerexpression and other commercially available cell separation andisolation kits (e.g., ISOCELL from Pierce, Rockford, Ill.). Particularsubpopulations of immune cells may be isolated through the use of beadsor other binding agents available in such kits specific to unique cellsurface markers. For example, MACS™ CD4+ and CD8+ MicroBeads may be usedto isolate CD4+ and CD8+ T-cells. The strategy of isolating andexpanding antigen-specific T cells as a therapeutic intervention forhuman disease has also been validated in clinical trials (Riddell etal., 1992; Walter et al., 1995; Heslop et al., 1996).

In some embodiments, isolation of the cells includes one or morepreparation and/or non-affinity-based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some embodiments, the blood cells collected from the subject arewashed, e.g., to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In someembodiments, the cells are washed with phosphate buffered saline (PBS).In some embodiments, the wash solution lacks calcium and/or magnesiumand/or many or all divalent cations. In some aspects, a washing step isaccomplished by a semi-automated “flow-through” centrifuge (for example,the Cobe 2991 cell processor, Baxter) according to the manufacturer'sinstructions. In some aspects, a washing step is accomplished bytangential flow filtration (TFF) according to the manufacturer'sinstructions. In some embodiments, the cells are resuspended in avariety of biocompatible buffers after washing, such as, for example,Ca²⁺/Mg²⁺ free PBS. In certain embodiments, components of a blood cellsample are removed and the cells directly resuspended in culture media.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner. Such separation steps can be based on positive selection, inwhich the cells having bound the reagents are retained for further use,and/or negative selection, in which the cells having not bound to theantibody or binding partner are retained. In some examples, bothfractions are retained for further use. In some aspects, negativeselection can be particularly useful where no antibody is available thatspecifically identifies a cell type in a heterogeneous population, suchthat separation is best carried out based on markers expressed by cellsother than the desired population.

In some embodiments, multiple rounds of separation steps are carriedout, where the positively or negatively selected fraction from one stepis subjected to another separation step, such as a subsequent positiveor negative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+,and/or CD45RO+ T cells, are isolated by positive or negative selectiontechniques. For example, CD3+ T cells can be expanded using CD3/CD28conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T CellExpander).

In some embodiments, isolation is carried out by enrichment for aparticular cell population by positive selection, or depletion of aparticular cell population, by negative selection. In some embodiments,positive or negative selection is accomplished by incubating cells withone or more antibodies or other binding agent that specifically bind toone or more surface markers expressed or expressed (marker+) at arelatively higher level on the positively or negatively selected cells,respectively.

In some embodiments, T cells are separated from a sample by negativeselection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4 or CD8 selection step is used to separate CD4+ helper and CD8+cytotoxic T cells. Such CD4+ and CD8+ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8+ cells are further enriched for or depleted ofnaive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memoryT (TCM) cells is carried outto increase efficacy, such as to improve long-term survival, expansion,and/or engraftment following administration, which in some aspects isparticularly robust in such sub-populations. See Terakura et al. (2012)Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ Tcells further enhances efficacy.

In embodiments, memory T cells are present in both CD62L+ and CD62L−subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L− CD8+ and/or CD62L+CD8 fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (TCM) cells isbased on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, and/or CD127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B. In some aspects, isolation of a CD8+ population enriched forTCM cells is carried out by depletion of cells expressing CD4, CD14,CD45RA, and positive selection or enrichment for cells expressing CD62L.In one aspect, enrichment for central memoryT (TCM) cells is carried outstarting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8+ cell population or subpopulation, also is used to generate theCD4+ cell population or sub-population, such that both the positive andnegative fractions from the CD4 based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In some embodiments, the enrichment for NK cells is based on positive orhigh surface expression of CD56 and CD16 and on the negative expressionof CD3 and/or optionally on the presence of NKp46 or NKp30 receptors.

In some aspects, the sample or composition of cells to be separated isincubated with small, magnetizable or magnetically responsive material,such as magnetically responsive particles or micro-particles, such asparamagnetic beads (e.g., such as Dynabeads or MACS beads). Themagnetically responsive material, e.g., particle, generally is directlyor indirectly attached to a binding partner, e.g., an antibody, thatspecifically binds to a molecule, e.g., surface marker, present on thecell, cells, or population of cells that it is desired to separate,e.g., that it is desired to negatively or positively select. In someembodiments, the magnetic particle or bead comprises a magneticallyresponsive material bound to a specific binding member, such as anantibody or other binding partner. There are many well-knownmagnetically responsive materials used in magnetic separation methods.Suitable magnetic particles include those described in Molday, U.S. Pat.No. 4,452,773, and in European Patent Specification EP 452342 B, whichare hereby incorporated by reference. Colloidal sized particles, such asthose described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby theantibodies or binding partners or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In some aspects, the sample is placed in a magnetic field, and thosecells having magnetically responsive or magnetizable particles attachedthereto will be attracted to the magnet and separated from the unlabeledcells. For positive selection, cells that are attracted to the magnetare retained; for negative selection, cells that are not attracted(unlabeled cells) are retained. In some aspects, a combination ofpositive and negative selection is performed during the same selectionstep, where the positive and negative fractions are retained and furtherprocessed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody- or otherbinding partner (e.g., streptavidin)-coated magnetic particles, areadded. In certain embodiments, streptavidin-coated magnetic particlesare used in conjunction with biotinylated primary or secondaryantibodies.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies,magnetizable particles or antibodies conjugated to cleavable linkers,etc. In some embodiments, the magnetizable particles are biodegradable.

In certain embodiments, the isolation or separation is carried out usinga system, device, or apparatus that carries out one or more of theisolation, cell preparation, separation, processing, incubation,culture, and/or formulation steps of the methods. In some aspects, thesystem is used to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inInternational Patent Application, Publication Number WO2009/072003, orUS 20110003380.

In some embodiments, a cell population described herein is collected andenriched (or depleted) via flow cytometry, in which cells stained formultiple cell surface markers are carried in a fluidic stream. In someembodiments, a cell population described herein is collected andenriched (or depleted) via preparative scale (FACS)-sorting. In certainembodiments, a cell population described herein is collected andenriched (or depleted) by use of microelectromechanical systems (MEMS)chips in combination with a FACS-based detection system (see, e.g., WO2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al.(2008) J Biophoton. l(5):355-376. In both cases, cells can be labeledwith multiple markers, allowing for the isolation of well-defined T cellsubsets at high purity. In some embodiments, the antibodies or bindingpartners are labeled with one or more detectable marker, to facilitateseparation for positive and/or negative selection. For example,separation may be based on binding to fluorescently labeled antibodies.

In some examples, separation of cells based on binding of antibodies orother binding partners specific for one or more cell surface markers arecarried in a fluidic stream, such as by fluorescence-activated cellsorting (FACS), including preparative scale (FACS) and/ormicroelectromechanical systems (MEMS) chips, e.g., in combination with aflow-cytometric detection system. Such methods allow for positive andnegative selection based on multiple markers simultaneously.

In some embodiments, the methods include density-based cell separationmethods, such as the preparation of white blood cells from peripheralblood by lysing the red blood cells and centrifugation through a Percollor Ficoll gradient.

In any of the aforementioned separation steps, the separation does notresult in 100% enrichment or removal of a particular cell population orcells expressing a particular marker. For example, positive selection ofor enrichment for cells of a particular type, such as those expressing amarker, refers to increasing the number or percentage of such cells, butdoes not result in a complete absence of cells not expressing themarker. Likewise, negative selection, removal, or depletion of cells ofa particular type, such as those expressing a marker, refers todecreasing the number or percentage of such cells, but does not resultin a complete removal of all such cells.

Alternatively, cells may be obtained through commercially available cellcultures, including but not limited to, for T cells, lines BCL2 (AAA)Jurkat (ATCC® CRL-2902™), BCL2 (S70A) Jurkat (ATCC® CRL-2900™), BCL2(S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat (ATCC® CRL-2899™), NeoJurkat (ATCC® CRL-2898™), TALL-104 (ATTC® CRL-11386); and, for NK cells,lines NK-92 (ATCC® CRL-2407™), NK-92MI (ATCC® CRL-2408™) Non-limitingexemplary sources for such commercially available cell lines include theAmerican Type Culture Collection, or ATCC, (http://www.atcc.org/) andthe German Collection of Microorganisms and Cell Cultures(https://www.dsmz.de/).

Cell Preparation and Expansion

Whether prior to or after genetic modification of the immune cells toexpress a desirable CAR, the cells can be activated and expanded usinggenerally known methods or from readily adapted method to the presentapplication such as those described in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7, 172,869; 7,232,566; 7, 175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application PublicationNo. 20060121005, Life Technologies Dynabeads® system activation andexpansion kits; BD Biosciences Phosflow™ activation kits, MiltenyiBiotec MACS™ activation/expansion kits, and other commercially availablecell kits specific to activation moieties of the relevant cell.Stimulation with the HLA-G antigen ex vivo can activate and expand theselected CAR expressing cell subpopulation. Alternatively, the cells maybe activated in vivo by interaction with HLA-G antigen.

The incubation and/or engineering may be carried out in a culturevessel, such as a unit, chamber, well, column, tube, tubing set, valve,vial, culture dish, bag, or other container for culture or cultivatingcells.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Insome embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor. The conditions caninclude one or more of particular media, temperature, oxygen content,carbon dioxide content, time, agents, e.g., nutrients, amino acids,antibiotics, ions, and/or stimulatory factors, such as cytokines,chemokines, antigens, binding partners, fusion proteins, recombinantsoluble receptors and any other agents designed to activate the cells.

In some embodiments, the immune cells of the invention can be expandedin vitro by co-culturing with tissue or cells. The cells can also beexpanded in vivo, for example in the subject's blood afteradministrating the cell into the subject.

Generally, the T cells of the invention can be expanded, for example, bycontact with an agent that stimulates a CD3 TCR complex and aco-stimulatory molecule on the surface of the T cells to create anactivation signal for the T cell. For example, chemicals such as calciumionophore A23187, phorbol 12-myristate β-acetate (PMA), or mitogeniclectins like phytohemagglutinin (PHA) can be used to create anactivation signal for the T cell.

In some embodiments, T cell populations may be stimulated in vitro bycontact with, for example, an anti-CD3 antibody, or antigen-bindingfragment thereof, or an anti-CD2 antibody immobilized on a surface, orby contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. In some embodiments, the T cellpopulations may be stimulated in vitro by contact with Muromonab-CD3(OKT3). For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be incubated with an anti-CD3antibody and an anti-CD28 antibody under conditions stimulatingproliferation of the T cells.

In some embodiments, the T cells are expanded by adding to theculture-initiating composition feeder cells, such as non-dividing PBMC,(e.g., such that the resulting population of cells contains at leastabout 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocytein the initial population to be expanded); and incubating the culture(e.g. for a time sufficient to expand the numbers of T cells). In someaspects, the non-dividing feeder cells can comprise gamma-irradiatedPBMC feeder cells. In some embodiments, the PBMC are irradiated withgamma rays in the range of about 3000 to 3600 rads to prevent celldivision.

In some aspects, the feeder cells are added to culture medium prior tothe addition of the populations of T cells.

In particular embodiments, co-stimulatory molecules are employed toenhance the activation, proliferation, and cytotoxicity of T cellsproduced by the CAR after antigen engagement. A co-stimulatory ligandcan include, but is not limited to, B7-1 (CD80), B7-2 (CD86), B7-H3,BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5, CD7, CD8a, CD8β, CD1a, LFA-1(CD11a/CD18), CD1b, CD1c, CD1d, CD18, CD19, CD19a, CD27, CD28, CD29,CD30, CD30L, CD40, CD40MICA, CD49a, CD49D, CD49f, CD69, CD70, CD83,CD84, CD96 (Tactile), CD 100 (SEMA4D), CD 103, OX40 (CD134), 4-1BB(CD137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD 162),DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1,CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), HLA-G, IA4, ICAM-1, IL2Rβ,IL2Rγ, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1,ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, MICB, NKG2C, NKG2D,NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PD-1, PD-L1, PD-L2, PSGL1,SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6,inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule(rCAM), lymphotoxin beta receptor,3 TR6, ILT3 and ILT4.

A co-stimulatory ligand also encompasses, inter alia, an antibody thatspecifically binds with a co-stimulatory molecule present on a T cell,such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83.

In some embodiments, NK cell populations can be expanded in vitro usinginterleukin-2 (IL-2) IL-15, IL-15/IL-15RA complex, IL-18 and IL-12.

Conditions appropriate for T cell and NK cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-Vivo 10, X-Vivo 15 and X-Vivo 20 (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,IL-7, GM-CSF, IL-10, IL-12, IL-2, IL-15, IL-18, IL-21, TGF , and TNF, orany other additives for the growth of cells known to the skilledartisan. In a preferred embodiment, T cells are stimulated in vitro byexposure to OKT3 and IL-2. Other additives for the growth of cellsinclude, but are not limited to, surfactant, Plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 10, X-Vivo 15,and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.

Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37 degrees Celsius) and atmosphere (e.g., air plus 5% CO₂). Tcells that have been exposed to varied stimulation times may exhibitdifferent characteristics.

In some embodiments, the preparation methods include steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. In some embodiments, the freeze andsubsequent thaw step removes granulocytes and, to some extent, monocytesin the cell population. In some embodiments, the cells are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This is then diluted 1:1 with media so that the finalconcentration of DMSO and HSA are 10% and 4%, respectively. The cellsare then frozen to -80° C. at a rate of 1 degree per minute and storedin the vapor phase of a liquid nitrogen storage tank.

In the inventive method the NK cells or T cells are preferably ex vivoexpanded for at least about 5 days, preferably not less than about 10days, more preferably not less than about 15 days and most preferablynot less than about 20 days before administration to the patient.

In another embodiment the NK cells or T cells have been expanded atleast about 100-fold, preferably at least about 200-fold, and morepreferably at least about 400-fold, preferably at least about 600-fold,more preferably at least about 1000 fold and even more preferably atleast about 1500 fold compared to day 0 of expansion, beforeadministration to a patient.

Cell Transduction and Selection

The nucleic acid construct according to the invention can be transducedinto immune cells to create an immune cell that expresses the anti-HLA-GCAR according to the invention. In certain embodiments, cells aretransduced to comprise at least one CAR of the present invention.

It is contemplated that the chimeric nucleic acid construct can beintroduced into the subject's own immune cells as naked DNA or in asuitable vector. Methods of stably transfecting immune cells,particularly T cell, by electroporation using naked DNA are known in theart. See e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers tothe DNA encoding a chimeric receptor of the present invention containedin a plasmid expression vector in proper orientation for expression.Advantageously, the use of naked DNA reduces the time required toproduce T cells expressing the chimeric receptor of the presentinvention. Physical methods for introducing a nucleic acid constructinto a host cell include calcium phosphate precipitation, lipofection,particle bombardment, microinjection, electroporation, and the like.

Alternatively, biological methods for introducing a polynucleotide ofinterest into a host cell include the use of DNA and RNA vectors. Avariety of viral vectors such as vector described hereabove can be usedto introduce the nucleic acid construct of the invention into immunecells. Suitable vectors for use in accordance with the method of thepresent invention do not replicate in the subject's immune cells.

In one embodiment, the nucleic acid construct encoding the CAR accordingto the invention is introduced into an immune cell by a viral vector,particularly a lentiviral vector as described hereabove.

Alternatively, chemical means for introducing a polynucleotide into ahost cell include colloidal dispersion systems, such as macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. An exemplary colloidal system for use as a delivery vehiclein vitro and in vivo is a liposome (e.g., an artificial membranevesicle).

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention. Methods of testing a CAR forthe ability to recognize target cells and for antigen specificity areknown in the art. For instance, Clay et al, J. Immunol., 163: 507-513(1999), teaches methods of measuring the release of cytokines (e.g.,interferon-γ, granulocyte/monocyte colony stimulating factor (GM-CSF),tumor necrosis factor a (TNF-α) or interleukin 2 (IL-2)). In addition,CAR function can be evaluated by measurement of cellular cytotoxicity,as described in Zhao et al, J. Immunol., 174: 4415-4423 (2005).

Once it is established that the transfected or transduced immune cell iscapable of expressing the chimeric receptor as a surface membraneprotein with the desired regulation and at a desired level, it can bedetermined whether the chimeric receptor is functional in the host cellto provide for the desired signal induction. Subsequently, thetransduced immune cells can be further reintroduced or administered tothe subject to activate anti-tumor responses in the subject. Tofacilitate administration, the transduced T cells according to theinvention can be made into a pharmaceutical composition or made into animplant appropriate for administration in vivo, with pharmaceuticallyacceptable carriers or diluents.

Once the cells expressing the CAR according to the invention areadministered to a subject, the biological activity of the engineeredcell populations and/or antibodies in some aspects is measured by any ofa number of known methods. Parameters to assess include specific bindingof an engineered or natural T cell or other immune cell to antigen, invivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.

In certain embodiments, the ability of the engineered cells to destroytarget cells can be measured using any suitable method known in the art,such as cytotoxicity assays described in, for example, Kochenderfer etal., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J.Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, thebiological activity of the cells also can be measured by assayingexpression and/or secretion of certain cytokines, such as GM-CSF, IL-3,MIP-1α, TNF-α, IL-10, IL-13, IFN-γ or IL-2.

In some aspects the biological activity is measured by assessingclinical outcome, such as reduction in tumor burden or load,stabilization of tumor, progression free survival, or overall survival.

Pharmaceutical or Veterinary Composition

The present invention also relates to a pharmaceutical or veterinarycomposition comprising the anti-HLA-G antibody or antibody fragment, theCAR, the nucleic acid construct, the vector and/or the cell as describedhereabove.

In a particular aspect, the present invention relates to apharmaceutical or veterinary composition comprising cells, preferablyimmune cells, comprising a CAR as described here above and/or comprisingthe nucleic acid construct encoding it as described hereabove. In oneaspect, the pharmaceutical or veterinary composition may comprise atleast two different populations of cells, a first one with cellscomprising a CAR specifically binding to β2M-associated HLA-G,preferably to both HLA-G1 and HLA-G5 and a second one with cellscomprising a CAR specifically binding to β2M-free HLA-G, preferably toboth HLA-G2 and HLA-G6 and/or to both HLA-G1/β2M-free andHLA-G5/β2M-free isoforms. In another aspect, the pharmaceutical orveterinary composition may comprise a population of cells comprising atleast two CARs, a first CAR specifically binding to β2M-associatedHLA-G, preferably to both HLA-G1 and HLA-G5 and a second CARspecifically binding to β2M-free HLA-G preferably to both HLA-G2 andHLA-G6 and/or to HLA-G1/β2M-free and/or HLA-G5/β2M-free isoforms. Inanother aspect, the pharmaceutical or veterinary composition maycomprise a population of cells comprising a bispecific CAR that allowsthe specific binding to β2M-associated HLA-G, preferably to both HLA-G1and HLA-G5 and to β2M-free HLA-G preferably to both HLA-G2 and HLA-G6and even more preferably to both HLA-G2 and HLA-G6 and/or to bothHLA-G1/β2M-free and HLA-G5/β2M-free isoforms.

In a further aspect, the pharmaceutical or veterinary composition maycomprise a first population of cells that express a CAR or at least twoCAR or a bispecific CAR as described hereabove targeting β2M-associatedHLA-G and/or β2M-free HLA-G isoforms, and a second population of cellsexpressing a CAR that does not recognize HLA-G but recognized an antigenknown to be a target of interest in CAR therapies such as anti-tumoraland/or anti-viral therapies. It will be understood that such secondpopulation of CAR expressing cells does not target HLA-G.

The present invention also relates to a pharmaceutical or veterinarycomposition containing a plurality of CAR-expressing cells of theinvention, such as T cells and/or NK cells. The pharmaceuticalcomposition may additionally comprise a pharmaceutically acceptablecarrier, diluent or excipient. The pharmaceutical composition mayoptionally comprise one or more further pharmaceutically activepolypeptides and/or compounds.

In one embodiment, the concentration of the cell expressing CARaccording to the invention which is included in the pharmaceutical orveterinary composition is at least 0.001 mg/ml, at least 0.1 mg/ml, atleast 0.5 mg/ml, at least 1 mg/ml, at least 5 mg/ml, at least 10 mg/ml,at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30mg/ml, at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml, at least50 mg/ml, at least 55 mg/ml, at least 60 mg/ml, at least 65 mg/ml, atleast 70 mg/ml, at least 75 mg/ml, at least 80 mg/ml, at least 85 mg/ml,at least 90 mg/ml, at least 95 mg/ml, at least 100 mg/ml, at least 105mg/ml, at least 110 mg/ml, at least 115 mg/ml, at least 120 mg/ml, atleast 125 mg/ml, at least 130 mg/ml, at least 135 mg/ml, at least 140mg/ml, at least 150 mg/ml, at least 175 mg/ml, at least 200 mg/ml, atleast 250 mg/ml, at least 275 mg/ml or at least 300 mg/ml.

In another embodiment, the concentration of the cell expressing CARaccording to the invention which is included in the pharmaceutical orveterinary composition is between 0.001-0.01 mg/ml, between 0.01-0.1mg/ml, between 0.1-1 mg/ml, between 1-10 mg/ml, between 10-50 mg/ml,between 50-100 mg/ml, between 50-150 mg/ml, between 50-200 mg/ml,between 50-250 mg/ml, between 50-300 mg/ml, between 100-200 mg/ml,between 100-300 mg/ml, or between 200-300 mg/ml.

In another embodiment, the pharmaceutical or veterinary compositioncomprises cells expressing the CAR according to the invention,particularly at least 100 cells, at least 200 cells, at least 400 cells,at least 500 cells, at least 700 cells, at least 1000 cells, at least1500 cells, at least 2000 cells, at least 3000 cells, at least 5000cells, at least 10 000 cells, at least 100 000 cells, at least 1 millioncells, at least 10 million cells or at least 100 million cellsexpressing the CAR according to the invention.

The pharmaceutical or veterinary composition according to the inventioncan be formulated for any conventional route of administration includinga topical, enteral, oral, parenteral, intranasal, intravenous,intramuscular, subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical or veterinary composition according tothe invention may be administered by enteral or parenteral route ofadministration. When administered parenterally, the pharmaceutical orveterinary composition according to the invention is preferablyadministered by intravenous route of administration. When administeredenterally, the pharmaceutical or veterinary composition according to theinvention is preferably administered by oral route of administration.

It will be understood by one skilled in the art that the formulations ofthe invention may be isotonic with human blood that is the formulationsof the invention have essentially the same osmotic pressure as humanblood. Such isotonic formulations generally have an osmotic pressurefrom about 250 mOSm to about 350 mOSm. Isotonicity can be measured by,for example, a vapor pressure or ice-freezing type osmometer. Tonicityof a formulation is adjusted by the use of tonicity modifiers. “Tonicitymodifiers” are those pharmaceutically acceptable inert substances thatcan be added to the formulation to provide an isotonicity of theformulation. Tonicity modifiers suitable for this invention include, butare not limited to, saccharides, salts and amino acids.

Compositions and formulations for parenteral, intrathecal, orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carder compounds andother pharmaceutically acceptable carriers or excipients.

The pharmaceutical or veterinary composition according to the inventionmay further comprise a pharmaceutically acceptable vehicle. Thus,additional aspects of the invention relate to compositions comprising acarrier and one or more of the products—e.g., a cell comprising ananti-HLA-G CAR, a nucleic acid, a vector, an anti-HLA-G antibody orantibody fragment—described in the embodiments disclosed herein. Theformulations can be sterilized and, if desired, mixed with auxiliaryagents such as carriers and excipients which do not deleteriouslyinteract with the products—e.g., a cell comprising an anti-HLA-G CAR, anucleic acid, a vector, an anti-HLA-G antibody or antibody fragment—ofthe formulation.

Preferably, the pharmaceutical or veterinary compositions of the presentinvention including but not limited to any one of the claimedcompositions may comprise CAR-expressing cells as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients as described hereafter.Desirably, a pharmaceutically acceptable form is employed which does notadversely affect the desired immune potentiating effects of recombinantcells according to the invention.

To facilitate administration, the transduced immune cells, preferably Tcells and/or NK cells transduced with the nucleic acid constructencoding the CAR according to the invention can be made into apharmaceutical composition for administration in vivo, with appropriatepharmaceutically acceptable carriers or diluents. The means of makingsuch a composition have been described in the art (see, for instance,Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins, 21st edition (2005).

Particularly, formulations comprising populations of CAR-expressingcells may include pharmaceutically acceptable excipient(s). Excipientsincluded in the formulations will have different purposes depending, forexample, on the CAR construct, the subpopulation of cells used, and themode of administration. The formulations comprising populations ofCAR-expressing cells will typically have been prepared and cultured inthe absence of any non-human components, such as animal serum (e.g.,bovine serum albumin).

The formulation or composition may also contain more than one activeingredient useful for the particular indication, disease, or conditionbeing treated with the binding molecules or cells, preferably those withactivities complementary to the binding molecule or cell, where therespective activities do not adversely affect one another. Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended.

The pharmaceutical or veterinary composition in some aspects can employtime-released, delayed release, and sustained release delivery systemssuch that the delivery of the composition occurs prior to, and withsufficient time to cause, sensitization of the site to be treated. Meansknown in the art can be used to prevent or minimize release andabsorption of the composition until it reaches the target tissue ororgan, or to ensure timed-release of the composition. Such systems canavoid repeated administrations of the composition, thereby increasingconvenience to the subject and the physician.

Subject, Regimen and Administration

The present invention relates to a pharmaceutical composition of thepresent invention or a CAR expressing cells of the present invention foruse as a medicament or for use for treating a disease or a disorder in asubject. It also relates to the use of a pharmaceutical composition ofthe present invention or a CAR expressing cells of the present inventionin the manufacture of a medicament for treating a disease or a disorderin a subject. Finally, it relates to a method for treating a disease ora disorder in a subject comprising administering a therapeuticallyeffective amount of a pharmaceutical composition of the presentinvention or a CAR expressing cells of the present invention to thesubject.

The human subject according to the invention may be a human at theprenatal stage, a new-born, a child, an infant, an adolescent or anadult, in particular an adult of at least 40 years old, preferably anadult of at least 50 years old, still more preferably an adult of atleast 60 years old, even more preferably an adult of at least 70 yearsold.

In some embodiments, the subject is a validated animal model fordisease, adoptive cell therapy, and/or for assessing toxic outcomes suchas cytokine release syndrome (CRS).

In some embodiments, the subject has persistent or relapsed disease,e.g., following treatment with another immunotherapy and/or othertherapy, including chemotherapy, radiation, and/or hematopoietic stemcell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments,the administration effectively treats the subject despite the subjecthaving become resistant to another therapy. In some embodiments, thesubject has not relapsed but is determined to be at risk for relapse,such as at a high risk of relapse, and thus the compound or compositionis administered prophylactically, e.g., to reduce the likelihood of orprevent relapse.

In some embodiments, the invention includes the administration of CARexpressing cells of the present invention or a composition containingCAR expressing cells to a subject, such as one having, at risk for, orsuspected of having a disease, condition or disorder. In someembodiments, the cells, and compositions are administered to a subjecthaving a particular disease or condition to be treated, e.g., viaadoptive cell therapy, such as adoptive T cell therapy. In someembodiments, the cells or compositions are administered to the subject,such as a subject having or at risk for a disease or condition. In someaspects, the methods thereby treat, e.g., ameliorate one or more symptomof the disease or condition. In a preferred embodiment, the subject hasbeen diagnosed with an immune disease, preferably a cancer. Diagnosticmethods of autoimmune disease or cancer are well known by the manskilled in the art.

In a particular embodiment, the subject has already received at leastone line of treatment, preferably several lines of treatment, prior tothe administration of immune cells according to the invention or of apharmaceutical or veterinary composition according to the invention.

Preferably, the treatment is administered regularly, preferably betweenevery day and every month, more preferably between every day and everytwo weeks, more preferably between every day and every week, even morepreferably the treatment is administered every day. In a particularembodiment, the treatment is administered several times a day,preferably 2 or 3 times a day, even more preferably 3 times a day.

The duration of treatment with the vector according to the invention,with the immune cells according to the invention or with apharmaceutical or veterinary composition according to the invention ispreferably comprised between 1 day and 20 weeks, more preferably between1 day and 10 weeks, still more preferably between 1 day and 4 weeks,even more preferably between 1 day and 2 weeks. In a particularembodiment, the duration of the treatment is of about 1 week.Alternatively, the treatment may last as long as the disease persists.

The form of the pharmaceutical or veterinary compositions, the route ofadministration and the dose of administration of immune cells accordingto the invention or of a pharmaceutical composition according to theinvention can be adjusted by the man skilled in the art according to thetype and severity of the infection, and to the patient, in particularits age, weight, sex, and general physical condition. The compositionsof the present invention may be administered in a number of waysdepending upon whether local or systemic treatment is desired.

In the case of adoptive cell therapy, methods for administration ofcells for adoptive cell therapy are known and may be used in connectionwith the provided methods and compositions. For example, adoptive T celltherapy methods are described, e.g., in US Patent ApplicationPublication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See,e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukaharaet al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al.(2013) PLoS ONE 8(4): e61338.

One skilled in the art will recognize that, although more than one routecan be used for administration, a particular route can provide a moreimmediate and more effective reaction than another route. For example,intradermal delivery may be advantageously used over inhalation for thetreatment of melanoma. Local or systemic delivery can be accomplished byadministration comprising application or instillation of the formulationinto body cavities, inhalation or insufflation of an aerosol, or byparenteral introduction, comprising intramuscular, intravenous,intraportal, intrahepatic, peritoneal, subcutaneous, or intradermaladministration.

Although systemic (intravenous, IV) injection is favored in clinicalapplications because of its ease of administration, several preclinicalstudies (Carpenito, et al. (2009) Proc. Natl. Acad. Sci. USA106:3360-3365; Song, et al. (2011) Cancer Res. 71:4617-4627;Parente-Pereira, et al. (2011) J. Clin. Immunol. 31:710-718) suggestthat the regional (intratumoral, IT or intraperitoneal, IP)administration of T cells may provide optimal therapeutic effects, whichmay be in part due to increased T cell trafficking to the tumor. Forexample, it has been shown that CAR T cells remain at the site ofinoculation with minimal systemic absorption when delivered via IP or ITroutes (Parente-Pereira, et al. (2011) J. Clin. Immunol. 31:710-718). Incontrast, after intravenous administration, CAR T cells initially reachthe lungs and then are redistributed to the spleen, liver, and lymphnodes. In addition, RNA CAR-electroporated T cells may be particularlysuitable for regional administration, due to the transient nature of theCAR expression on the T cells (Zhao, et al. (2010) Cancer Res.70:9053-9061). Furthermore, clinical studies have shown the feasibilityand safety of both the intratumoral and intraperitoneal injection of Tcells (Canevari, et al. (1995) J. Natl. Cancer Inst. 87:1463-1469;Duval, et al. (2006) Clin. Cancer Res. 12:1229-1236). Overall, a localroute of administration of the recombinant T cells may provide theoptimal therapeutic effect and decrease the potential for the“on-target, off-organ” toxicity. Accordingly, in one embodiment, the CARexpressing cells according to the invention are administered locally,preferably by intra-tumoral and intraperitoneal injection.

The pharmaceutical composition in some embodiments contains the CARcells in amounts effective to treat or prevent the disease or condition,such as a therapeutically effective or prophylactically effectiveamount. Therapeutic or prophylactic efficacy in some embodiments ismonitored by periodic assessment of treated subjects. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful and can bedetermined. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition. The amount of immune cells according to the invention or ofa pharmaceutical composition according to the invention to beadministered can be determined by standard procedure well known by thoseof ordinary skills in the art. Physiological data of the patient (e.g.age, size, weight and health and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment and the nature ofthe effect desired) and the routes of administration have to be takeninto account to determine the appropriate dosage, so as atherapeutically effective amount will be administered to the patient.Particularly, the appropriate dosages and dosing schedule can be basedon clinical trials or well-established cell-based therapies (see, e.g.,Topalian & Rosenberg (1987) Acta Haematol. 78 Suppl 1:75-6; U.S. Pat.No. 4,690,915) or an alternate continuous infusion strategy can beemployed.

In some embodiments, an effective amount or number of cells orpharmaceutical composition comprising those cells are administratedparenterally. In some embodiments, administration can be an intravenousadministration. In some embodiments, administration can be directly doneby injection within a tumor.

In certain embodiments, in the context of genetically engineered cellsexpressing the CARs, a subject is administered the range of about onemillion to about 100 billion cells, such as, e.g., 1 million to about 50billion cells (e.g., about 5 million cells, about 25 million cells,about 500 million cells, about 1 billion cells, about 5 billion cells,about 20 billion cells, about 30 billion cells, about 40 billion cells,or a range defined by any two of the foregoing values), such as about 10million to about 100 billion cells (e.g., about 20 million cells, about30 million cells, about 40 million cells, about 60 million cells, about70 million cells, about 80 million cells, about 90 million cells, about10 billion cells, about 25 billion cells, about 50 billion cells, about75 billion cells, about 90 billion cells, or a range defined by any twoof the foregoing values), and in some cases about 100 million cells toabout 50 billion cells (e.g., about 120 million cells, about 250 millioncells, about 350 million cells, about 450 million cells, about 650million cells, about 800 million cells, about 900 million cells, about 3billion cells, about 30 billion cells, about 45 billion cells) or anyvalue in between these ranges, and/or such a number of cells perkilogram of body weight of the subject. For example, in some embodimentsthe administration of the cells or population of cells can compriseadministration of about 10³ to about 10⁹ cells per kg body weightincluding all integer values of cell numbers within those ranges, forexample, the cell compositions of the present invention can beadministered in a dose, or dosages, where each dose comprises at least10 cells/kg body weight, at least 100 cells/kg body weight; at least1000 cells/kg body weight; at least 10,000 cells; at least 100,000cells; at least 1 million cells; at least 10 million cells; at least 100million cells; at least 1 billion cells or at least 10 billion cells/kgbody weight.

Particularly, a sufficient number of the transduced immune cells will beintroduced so as to achieve the desired therapeutic response. Desirablyan effective amount or sufficient number of the isolated transducedcells is present in the composition and introduced into the subject suchthat long-term, specific, anti-tumor or anti-infectious agent responsesare established to reduce the size or regrowth of a tumor or growth ofan infectious agent than would otherwise result in the absence of suchtreatment. Desirably, the amount of transduced immune cells, preferablyT cells, reintroduced into the subject causes a 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size whencompared to otherwise same conditions, wherein the transduced immunecells are not present.

A composition of the invention can be provided in unit dosage formwherein each dosage unit, e.g. an injection, contains a predeterminedamount of the composition, alone or in appropriate combination withother active agents. The term unit dosage form, as used herein, refersto physically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of thecomposition of the invention, alone or in combination with other activeagents, calculated in an amount sufficient to produce the desiredeffect, in association with a pharmaceutically acceptable diluent,carrier, or vehicle, where appropriate. The specifications for the novelunit dosage forms of the invention depend on the particularpharmacodynamics associated with the pharmaceutical composition in theparticular subject.

The cells or population of cells can be administrated in one or moredoses. In some embodiments, said effective amount or number of cells canbe administrated as a single dose. In some embodiments, said effectiveamount or number of cells can be administrated as more than one doseover a period time. Timing of administration is within the judgment ofmanaging physician and depends on the clinical condition of the patient.While individual needs vary, determination of optimal ranges ofeffective amounts of a given cell type for a particular disease orconditions within the skill of the art. An effective amount means anamount which provides a therapeutic or prophylactic benefit.

For purposes of the invention, the amount or dose of the inventive CARmaterial administered should be sufficient to generate a therapeutic orprophylactic response in the subject over a reasonable time frame. Forexample, the dose of the inventive CAR material should be sufficient tobind to antigen, e.g. HLA-G isoform(s), or detect, treat or preventdisease in a period of from about 2 hours or longer, e.g., about 12 toabout 24 or more hours, from the time of administration. In certainembodiments, the time period could be even longer. The dose will bedetermined by the efficacy of the particular inventive CAR material andthe condition of the subject, as well as the body weight of the subjectto be treated.

For purposes of the invention, an assay, which comprises, for example,comparing the extent to which target cells are lysed or IFN-γ issecreted by T cells expressing the inventive CAR, polypeptide, orprotein upon administration of a given dose of such T cells to a mammal,among a set of mammals of which is each given a different dose of the Tcells, could be used to determine a starting dose to be administered toa mammal. The extent to which target cells are lysed or IFN-γ issecreted upon administration of a certain dose can be assayed by methodsknown in the art.

Uses

Use in the Treatment of Disease or Disorder and in Combination Therapy

In one aspect, the disease or disorder to be treated is a conditionselected from a proliferative disease or disorder, preferably cancer; aninfectious disease or disorder, preferably a viral infection; aninflammatory disease or disorder; and an immune disease or disorder,preferably autoimmunity or autoimmune diseases, allergies andgraft-vs-host rejection. In some embodiments, the condition may becancer.

The present invention relates to the use of an antibody, a cell, anucleic acid construct, a vector and/or a pharmaceutical compositionaccording to the invention for interfering or neutralizing the immunedown-regulation due to HLA-G proteins in a host in need thereof.

In particular, the cell, the nucleic acid construct, the vector and/orthe pharmaceutical composition according to the invention areparticularly suitable for treatment of viral infections such as forexample HIV-1, hepatitis B virus, and hepatitis C virus infections.

In particular, the cell, the nucleic acid construct, the vector and/orthe pharmaceutical composition according to the invention areparticularly suited for treatment of cancer, particularly of solidtumors or hematopoietic cancer, even more preferably when theavailability of good selective single targets is limited.

The immune system can specifically identify and eliminate tumor cellsbased on their expression of tumor-specific antigens or moleculesinduced during malignant cell transformation. This process is referredto as tumor immune surveillance. Despite tumor immune surveillance,tumors can still develop in the presence of a functioning immune system.This occurs through tumor immunoediting, a process that comprises threemajor phases: 1) the elimination phase in which most immunogenic tumorcells are eliminated by cytotoxic T and NK cells; 2) the equilibriumphase in which tumor cells with reduced immunogenicity are selected; and3) the escape phase in which variants that no longer respond to the hostimmune system are maintained (Urosevic and Dummer, 2008). HLA-G isinvolved in every phase of tumor immuno-editing by decreasing theelimination of tumor cells, by inhibiting the cytotoxic function of Tand NK cells, and by trogocytosis, (i.e. the intercell transference ofviable HLA-G molecules), which renders competent cytotoxic cellsunresponsive to tumor antigens (LeMaoult et al., 2007; Caumartin et al.,2007). Therefore, the chimeric constructs of the present invention findapplication in subjects having or suspected of having a disease,disorder, or a particular condition, particularly subjects having orsuspected of having a cancer. Particularly the chimeric constructs ofthe present invention find application in subjects having or suspectedof having a cancer thereby reducing the size of a tumor or preventingthe growth or re-growth of a tumor in these subjects or preventing theinduction of an immunosuppressive microenvironment.

Accordingly, the present invention also relates to methods forinhibiting the growth of a tumor in a subject in need thereof and/or fortreating a cancer patient in need thereof. The tumor may be a solidtumor, or a liquid tumor. In some embodiments, the tumor or cancerexpresses or overexpresses HLA-G. In certain embodiments, these methodscomprise, or alternatively consist essentially of, or yet furtherconsist of, administering to the subject or patient an effective amountof the isolated cell. In still further embodiments, the cell expressinga CAR according to the invention is a T cell or an NK cell. The isolatedcell may be allogeneic or autologous to the subject or patient beingtreated. In a further aspect, the tumor expresses or overexpresses HLA-Gantigen and the subject has been selected for the therapy by adiagnostic.

In one embodiment, the present invention relates to a method forreducing growth or preventing tumor formation in a subject byintroducing a chimeric construct of the present invention into an immunecell, preferably a T cell or a NK cell, of the subject and reintroducinginto the subject the transformed immune cell, thereby expressing the CARaccording to the invention and effecting anti-tumor responses to reduceor eliminate tumors in the subject. The step of delivering the nucleicacid construct to the subject generally involves introducing a nucleicacid construct of the invention into an isolated immune cell (e.g., anautologous immune cell isolated from PBMC or immune cells derived froman allogeneic third party-derived immune cell donor) and introducinginto the subject the transformed immune cell, thereby effectingantitumor responses to reduce or eliminate tumors in the subject, as inan adoptive T cell therapy method. For example, the immune cell maycomprise a T cell and the subject is suffering from, or is believed tobe suffering from, or is diagnosed as having tumor or cancer, e.g., aHLA-G expressing cancer.

For example, the anti-HLA-G CAR molecules encoded by exemplary nucleicacid constructs of the present invention may be administered to thesubject in the form of a recombinant immune cell engineered to expressthe anti-HLA-G CAR molecule.

CAR expressing cells according to invention and obtained by the methodsdescribed above, or cell lines derived from such cells, can be used as amedicament in the treatment of a disease, disorder, or condition in asubject. In some embodiments, such a medicament can be used for treatingcancer.

In some embodiments, administering the treatment to the subject maycomprise adoptive cell therapy (ACT) using immune cells harvested fromthe subject or from one or more donors. Accordingly, the cells can becells that are xenogeneic, allogeneic or autologous to the subject.Generally, the cells are autologous to the subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by autologous transfer, inwhich the cells are isolated and/or otherwise prepared from the subjectwho is to receive the cell therapy, or from a sample derived from such asubject. Thus, in some aspects, the cells are derived from a subject,e.g., patient, in need of a treatment and the cells, following isolationand processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by allogeneic transfer, inwhich the cells are isolated and/or otherwise prepared from a subjectother than a subject who is to receive or who ultimately receives thecell therapy, e.g., a first subject. In such embodiments, the cells thenare administered to a different subject, e.g., a second subject, of thesame species. In some embodiments, the first and second subjects aregenetically identical. In some embodiments, the first and secondsubjects are genetically similar. In some embodiments, the secondsubject expresses the same HLA class or supertype as the first subject.The cells of the present invention may be capable of killing targetcells, such as cancer cells. The target cell may be recognizable by adefined pattern of antigen expression, for example the expression ofantigen A or antigen B.

In some embodiments, ACT may comprise isolating primary immune cellsfrom the subject or from one or more donors, transducing the primaryimmune cells with the nucleic acid construct or constructs of any of theforegoing embodiments, expressing the CAR in the transduced primaryimmune cells, and delivering the transduced immune cells into thesubject. ACT may further comprise stimulating and/or expanding theimmune cells prior to delivering the transduced immune cells to thesubject.

For example, in some embodiments, ACT may comprise harvesting autologousor allogeneic T cells and transducing these T cells with one or morenucleic acid constructs, so that the T cells express a CAR mediatingpro-inflammatory cytokine expression, and then infusing the cells into asubject in need thereof.

The invention also provides a method for treating cancer comprisingdelivering to a subject in need thereof an effective amount of thenucleic acid construct, a vector or vectors, or a transduced immune cellor pharmaceutical composition according to any of the foregoingembodiments, thereby treating the cancer. In some embodiments, thetreatment of cancer may be measured by a decrease in tumor cell burdenor by an increase in survival.

The invention additionally provides a method of immune therapycomprising administering to a subject a therapeutically effective amountof a nucleic acid construct or constructs, a vector or vectors, arecombinant cell or a pharmaceutical composition according to any of theforegoing embodiments. Treatment with the cells of the invention mayhelp prevent the escape or release of tumor cells which often occurswith standard approaches.

In certain embodiments, CAR expressing cells are modified in any numberof ways, such that their therapeutic or prophylactic efficacy isincreased. For example, the CAR may be conjugated either directly orindirectly through a linker to a targeting moiety. The practice ofconjugating compounds, e.g., the CAR, to targeting moieties is known inthe art. See, for instance, Wadhwa et al., J. Drug Targeting 1995; 3(2):111-127, and U.S. Pat. No. 5,087,616. Particularly, the presentinvention includes a type of cellular therapy where isolated cells aregenetically modified to express CARs and the CAR cell is infused into asubject in need thereof. Such administration can promote activation ofthe cells (e.g., T cell activation) in a target-specific manner, suchthat the cells of the disease or disorder are targeted for destruction.In the case where the cell is a T cell, CAR T cells, unlike antibodytherapies, are able to replicate in vivo resulting in long-termpersistence that may lead to sustained control of targeted diseases,disorders, or conditions.

The CAR expressing cells as disclosed herein may be administered eitheralone or in combination with diluents, known anti-cancer therapeutics,and/or with other components such as cytokines or other cell populationsthat are immunostimulatory. They may be administered as a first linetherapy, a second line therapy, a third line therapy, or furthertherapy.

In some embodiments, the cells expressing CAR are administered as partof a combination treatment, such as simultaneously with or sequentiallywith, in any order, another therapeutic intervention, such as anantibody or engineered cell or receptor or agent, such as a cytotoxic ortherapeutic agent. The cells or antibodies in some embodiments areco-administered with one or more additional therapeutic agents or inconnection with another therapeutic intervention, either simultaneouslyor sequentially in any order.

In some contexts, the cells are co-administered with another therapysufficiently close in time such that the cell populations enhance theeffect of one or more additional therapeutic agents, or vice versa. Insome embodiments, the cells or antibodies are administered prior to theone or more additional therapeutic agents. In some embodiments, thecells or antibodies are administered after to the one or more additionaltherapeutic agents, such as anti-cancer agents. An “anti-cancer” agentis capable of negatively affecting cancer in a subject, for example, bykilling cancer cells, inducing apoptosis in cancer cells, reducing thegrowth rate of cancer cells, reducing the incidence or number ofmetastases, reducing tumor size, inhibiting tumor growth, reducing theblood supply to a tumor or cancer cells, promoting an immune responseagainst cancer cells or a tumor, preventing or inhibiting theprogression of cancer, or increasing the lifespan of a subject withcancer. More generally, these other compositions would be provided in acombined amount effective to kill or inhibit proliferation of the cell.This process may involve contacting the cancer cells with the cellexpressing the CAR according to the invention and the agent(s) ormultiple factor(s) at the same time. This may be achieved by contactingthe cell with a single composition or pharmacological formulation thatincludes both agents, or by contacting the cell with two distinctcompositions or formulations, at the same time, wherein one compositionincludes the expression construct and the other includes the secondagent(s).

In a particular embodiment, the cells expressing a CAR against HLA-Gisoforms according to the invention are administered as part of acombination treatment such as simultaneously with or sequentially with,in any order, with other CAR expressing cells that does not recognizeHLA-G but are known to be useful in other CAR therapies such asanti-tumoral and/or anti-viral CAR therapies. Such CAR expressing cellstargets an antigen involved in a disease, preferably such as cancer orviral infection, preferably an antigen targeted in cancer therapies orin viral therapies. It will be understood that such antigen is notHLA-G.

In the context of the present invention, it is contemplated that celltherapy could be used similarly in conjunction with chemotherapeutic,radiotherapeutic, or immunotherapeutic intervention, as well aspro-apoptotic or cell cycle regulating agents such as immune checkpointinhibitor.

Alternatively, the present inventive therapy may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and present invention are appliedseparately to the individual, one would generally ensure that asignificant period of time did not expire between the times of eachdelivery, such that the agent and inventive therapy would still be ableto exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several week (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It is expected that the treatment cycles would be repeated as necessary.It also is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the inventivecell therapy.

Targeted Cancers

HLA-G is aberrantly expressed in many human solid malignant tumors insitu and malignant hematopoietic diseases including breast, ovarian,clear renal cell, colorectal, gastric, esophageal, lung, andhepatocellular cancers, as well as acute myeloid leukemia and chroniclymphocytic leukemia (B-CLL). The aberrant expression of HLA-G inmalignant neoplasm is significantly correlated with poor clinicaloutcome of patients with colorectal cancer (CRC), gastric cancer (GC),non-small cell lung cancer (NSCLC), esophageal squamous cell cancer(ESCC), breast cancer, hepatocellular cancers, and B-CLL. Furthermore,serum soluble HLA-G is increased in various types of cancer patients(including patients with melanoma, acute leukemia, multiple myeloma,neuroblastoma, lymphoproliferative disorders, breast or ovarian cancer,non-small cell lung cancer, esophageal cancer, colorectal cancer;gastric cancer and hepatocellular carcinoma), when compared to normalhealthy controls or benign disease cases.

Cancers that may be treated by the CAR expressing cell, the nucleic acidconstruct, the vector or the pharmaceutical composition according to theinvention include tumors that are not vascularized, or not yetsubstantially vascularized, as well as vascularized tumors.

The CAR expressing cell, the nucleic acid construct, the vector or thepharmaceutical composition according to the invention may be used totreat cancers of the oral cavity and pharynx which includes cancer ofthe tongue, mouth and pharynx; cancers of the digestive system whichincludes esophageal, gastric and colorectal cancers; cancers of theliver and biliary tree which includes hepatocellular carcinomas andcholangiocarcinomas; cancers of the respiratory system which includesbronchogenic cancers, lung cancers and cancers of the larynx; cancers ofbone and joints which includes osteosarcoma; cancers of the skin whichincludes melanoma; breast cancer; cancers of the genital tract whichinclude uterine, endometrium, ovarian and cervical cancer in women,prostate and testicular cancer in men; cancers of the renal tract whichinclude renal cell carcinoma and transitional cell carcinomas of theutterers or bladder; gastrointestinal stromal tumor, pancreas cancers,kidney cancers, colon cancers, cervix cancer, brain cancers includinggliomas, glioblastoma multiform and medullobastomas; cancers of theendocrine system including thyroid cancer, adrenal carcinoma and cancersassociated with multiple endocrine neoplasm syndromes; lymphomasincluding Hodgkin's lymphoma and non-Hodgkin lymphoma; B-cell lymphoma,monocytic lymphoma, marginal zone lymphoma, Burkitt's lymphoma, T and Blymphomas, Multiple Myeloma and plasmacytomas; leukaemias both acute andchronic, prohemocytic leukemia, acute non-lymphoblastic leukemia (ANLL),acute lymphoblastic leukemia (ALL), erythroleukemia, myeloid or lymphoidleukemia; and cancers of other and unspecified sites includingneuroblastoma. Preferably, the cancer is selected from the group ofRenal cell carcinoma (RCC), melanoma, kidney cancer and bladder cancer.

The cancers may comprise non solid tumors (such as hematological tumors,for example, leukemia and lymphoma) or may comprise solid tumors. Asused herein, “solid tumor” is an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors can be benign ormalignant. Different types of solid tumors are named for the type ofcells that form them (such as sarcomas, carcinomas, and lymphomas).

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma,and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNStumors (such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

In some embodiments, the cancer cells express or over express HLA-G. Ina particular embodiment, the cancer cells express or overexpress one tosix, preferably two to five HLA-G isoform(s) selected from HLA-G1,HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6 and HLA-G7, preferably fromHLA-G1, HLA-G2, HLA-G5 or HLA-G6, and more preferably from HLA-G1 orHLA-G2.

Preferably, the cancer cells express or overexpress HLA-G1 and/orHLA-G5; or the cancer cells express or overexpress HLA-G2 and/or HLA-G6.Preferably, when such cancer cells express these particular HLA-Gisoforms, the CAR according to the invention specifically binds toHLA-G1 and HLA-G5 or to HLA-G2 and HLA-G6, respectively.

Use in Diagnostic and Prognostic

The anti-HLA-G monoclonal antibodies or scFv disclosed herein are alsouseful in diagnostic and prognostic methods. As such, the presentinvention relates to the use of the antibodies disclosed herein in thediagnosis of HLA-G-related medical conditions in a subject.

The monoclonal antibodies or scFv disclosed herein are useful in methodsknown in the art relating to the localization and/or quantitation of aHLA-G polypeptide (e.g., for use in measuring levels of the HLA-Gpolypeptide within appropriate physiological samples, for use indiagnostic methods, for use in imaging the polypeptide, and the like).The monoclonal antibodies or scFv disclosed herein are useful inisolating a HLA-G polypeptide by standard techniques, such as westernblotting, affinity chromatography methods for isolating cells or forflow cytometry-based cellular analysis or cell sorting orimmunoprecipitation. A HLA-G antibody disclosed herein can facilitatethe purification of natural HLA-G polypeptides from biological samples,e.g., mammalian sera or cells as well as recombinantly-produced HLA-Gpolypeptides expressed in a host system. Moreover, HLA-G monoclonalantibodies or scFv can be used to detect a HLA-G polypeptide (e.g., inplasma, a cellular lysate or cell supernatant) in order to evaluate theabundance and pattern of expression of the polypeptide. The HLA-Gantibodies disclosed herein can be used diagnostically to monitor HLA-Glevels in tissue as part of a clinical testing procedure, e.g., todetermine the efficacy of a given treatment regimen. The detection canbe facilitated by coupling (i.e., physically linking) the HLA-Gantibodies disclosed herein to a detectable substance so as the HLA-Gantibodies or fragments thereof are detectably labeled. The term“labeled”, with regard to the antibody is intended to encompass directlabeling of the antibody by coupling (i.e., physically linking) adetectable substance to the antibody, as well as indirect labeling ofthe antibody by reactivity with another compound that is directlylabeled. Non-limiting examples of indirect labeling include detection ofa primary antibody using a fluorescently-labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently-labeled streptavidin.

The detection method of the present disclosure can be used to detectexpression levels of HLA-G polypeptides in a biological sample in vitroas well as in vivo. In vitro techniques for detection of HLA-Gpolypeptides include enzyme linked immunosorbent assays (ELISAs),Western blots, flow cytometry, immunoprecipitations, radioimmunoassay,and immunofluorescence (e.g., IHC). Furthermore, in vivo techniques fordetection of HLA-G polypeptides include introducing into a subject alabeled anti-HLA-G antibody. By way of example only, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

In some aspects, HLA-G antibodies containing structural modificationsthat facilitate rapid binding and cell uptake and/or slow release areuseful in in vivo imaging detection methods. In some aspects, the HLA-Gantibody contains a deletion in the CH2 constant heavy chain region ofthe antibody to facilitate rapid binding and cell uptake and/or slowrelease. In some aspects, a Fab fragment is used to facilitate rapidbinding and cell uptake and/or slow release. In some aspects, a F(ab)′2fragment is used to facilitate rapid binding and cell uptake and/or slowrelease.

Accordingly, the present invention also provides prognostic (orpredictive) assays for determining whether a subject is at risk ofdeveloping a medical disease or condition associated with increasedHLA-G polypeptide expression or activity (e.g., detection of aprecancerous cell). Such assays can be used for prognostic or predictivepurpose to thereby prophylactically treat an individual prior to theonset of a medical disease or condition characterized by or associatedwith HLA-G polypeptide expression.

Another aspect of the present disclosure provides methods fordetermining HLA-G expression in a subject to thereby select appropriatetherapeutic or prophylactic compounds for that subject.

Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing for developing cancer and/orsolid tumors. Thus, the present disclosure provides a method foridentifying a disease or condition associated with increased HLA-Gisoform(s) expression levels in which a test sample is obtained from asubject and the HLA-G isoform(s) detected, wherein the presence ofincreased levels of HLA-G polypeptides compared to a control sample ispredictive for a subject having or at risk of developing a disease orcondition associated with increased HLA-G isoform(s) expression levels.In some aspects, the disease or condition associated with increasedHLA-G isoform(s) expression levels is selected from the group consistingof for developing cancer and/or solid tumors.

In another embodiment, the present disclosure provides methods fordetermining whether a subject can be effectively treated with a compoundfor a disorder or condition associated with increased HLA-G expressionwherein a biological sample is obtained from the subject and the HLA-Gisoform(s) is/are detected using the HLA-G antibody or ScFv as describedabove. The expression level of the HLA-G polypeptide in the biologicalsample obtained from the subject is determined and compared with theHLA-G expression levels found in a biological sample obtained from asubject who is free of the disease. Elevated levels of the HLA-G in thesample obtained from the subject suspected of having the disease orcondition compared with the sample obtained from the healthy subject isindicative of the HLA-G-associated disease or condition in the subjectbeing tested.

There are a number of disease states in which the elevated expressionlevel of HLA-G isoform(s) is known to be indicative of whether a subjectwith the disease is likely to respond to a particular type of therapy ortreatment. Thus, the method of detecting HLA-G isoform(s) in abiological sample can be used as a method of prognosis, e.g., toevaluate the likelihood that the subject will respond to the therapy ortreatment.

Further aspects of the present disclosure relate to methods fordetermining if a patient is likely to respond or is not likely to HLA-GCAR therapy. In specific embodiments, this method comprises contacting atumor sample isolated from the patient with an effective amount of anHLA-G antibody and detecting the presence of any antibody bound to thetumor sample. In further embodiments, the presence of antibody bound tothe tumor sample indicates that the patient is likely to respond to theHLA-G CAR therapy and the absence of antibody bound to the tumor sampleindicates that the patient is not likely to respond to the HLA-Gtherapy. In some embodiments, the method comprises the additional stepof administering an effective amount of the HLA-G CAR therapy to apatient that is determined likely to respond to the HLA-G CAR therapy.

Further aspects of the present disclosure relate to methods fordetermining if a patient is likely to respond or is not likely to HLA-GCAR therapy depending on the isoform(s) that is/are expressed by thetumor, particularly selected from HLA-G1, HLA-G2, HLA-G3, HLA-G4,HLA-G5, HLA-G6 and HLA-G7, preferably from HLA-G1, HLA-G2, HLA-G5 orHLA-G6, and more preferably from HLA-G1 or HLA-G2, even more preferablyHLA-G1 and G5 or HLAG-2 and G6. The identification of HLA-G expressedisoform(s) prior to treatment allowed the selection of the most suitableCAR that specifically binds one to six, preferably two to five HLA-Gisoform(s) selected from HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6and HLA-G7, preferably from HLA-G1, HLA-G2, HLA-G5 or HLA-G6, and morepreferably from HLA-G1 or HLA-G2, even more preferably binds HLA-G1 andHLA-G5 isoforms or both HLA-G2 and HLA-G6 isoforms for use in anefficient treatment such as cell therapy.

Kits

Any of the compositions described herein may be included in a kitprovided by the present invention. The kits will thus include, insuitable container means, recombinant/engineered cells of the presentinvention, and/or vectors encoding the nucleic acid constructs of thepresent invention, and/or nucleic acid constructs or related reagents ofthe present invention. In some embodiments, the kit further includes anadditional agent for treating cancer or an infectious disease, and theadditional agent may be combined with the nucleic acid construct(s) orcells, or other components of the kit of the present invention or may beprovided separately in the kit. In some embodiments, means of taking asample from an individual and/or of assaying the sample may be providedin the kit. In certain embodiments the kit includes cells, buffers, cellmedia, vectors, primers, restriction enzymes, salts, and so forth, forexample. The kits may also comprise means for containing a sterile,pharmaceutically acceptable buffer and/or other diluent.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.Such containers may include injection or blow-molded plastic containersinto which the desired vials are retained. The kits of the presentinvention also will typically include a means for containing thecomponents in close confinement for commercial sale. Such containers mayinclude injection or blow molded plastic containers into which thedesired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The compositions may alsobe formulated into a syringe compatible composition. In which case, thecontainer means may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit. However, the components ofthe kit may be provided as dried powder(s). When reagents and/orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

In particular embodiments of the invention, cells that are to be usedfor cell therapy are provided in a kit, and in some cases the cells areessentially the sole component of the kit. The kit may comprise reagentsand materials to make the desired cell. In specific embodiments, thereagents and materials include primers for amplifying desired sequences,nucleotides, suitable buffers or buffer reagents, salt, and so forth,and in some cases the reagents include vectors and/or DNA that encodes aCAR as described herein and/or regulatory elements therefor.

In particular embodiments, there are one or more apparatuses in the kitsuitable for extracting one or more samples from an individual. Theapparatus may be a syringe, scalpel, and so forth.

In some cases of the invention, the kit, in addition to cell therapyembodiments, also includes a second cancer therapy, such as chemotherapyand/or immunotherapy, for example. The kit(s) may be tailored to aparticular cancer for an individual and comprise respective secondcancer therapies for the individual as described hereabove.

EXAMPLES

Results

Anti-HLA-G Antibody Paratope Specificity

It has been previously described that different isoforms of HLA-G can beexpressed containing from one to three globular domains, associated ornot to β2M and a peptide. Different specific anti-HLA-G antibodies hadbeen previously generated against different regions or “epitopes” ofHLA-G. Specificity of anti-HLA-G monoclonal antibodies was previouslydetermined against different isoforms of HLA-G. Briefly, as shown onFIG. 2A, 15E7 antibody presents a high affinity for HLA-G1/5 β2M-freeand HLA-G6, demonstrating that this Mab is specific for HLA-G isoformsthat are not associated to β2M. As shown on FIG. 2B, K562 HLA-G1ppresents a higher proportion of HLA-G β2M-free than K562 HLA-G1Lv afterstaining with the 15E7 monoclonal antibody in comparison to the MEM-G/09monoclonal antibody, specific for HLA-G β2M-associated isoforms (datanot shown). Similarly, 15E7 specificity was evaluated against Jurkat wt,Jurkat HLA-G1Lv and Jeg-3 cell lines which demonstrated that the 15E7monoclonal antibody presents high affinity for HLA-G β2M-free isoforms,as shown on FIG. 2B.

Specificity of the LFTT-1 monoclonal antibody was determined performingthe same experiments. Contrary to 15E7 antibody, LFTT-1 antibody ishighly specific for HLA-G β2M-associated isoforms and stained K562HLA-G1Lv, Jurkat HLA-G1Lv and Jeg-3 cell lines, as shown on FIG. 2C.

CARs Constructions Validation

Two different chimeric antigen receptors were generated against HLA-Gbased on the 15E7 and LFTT-1 monoclonal antibodies paratopes. CARsdesign corresponded to the 3^(rd) generation CARs already described(Pule et al. 2005 Molecular Therapy) constituted of: a signal peptide,the scFv of either 15E7 or LFTT-1, a spacer domain that comprises orconsists of (i) a CD8a or CD28 hinge, (ii) a human IgG4 hinge domain,(iii) a human IgG4 hinge domain and a CH3 human IgG4 domain or (iv) amutated CH2 human IgG4 domain, a human IgG4 hinge domain and a CH3 humanIgG4 hinge domain., a CD28 transmembrane region, and two co-activationsegments: 4-1BB and CD3ζ. FIG. 3 summarizes the schematic design of theCAR structures based on 15E7 and LFTT-1 with CD8a or CD28 hinge domainand FIG. 4 summarizes the schematic design of the CAR structures basedon 15E7 and LFTT-1 with the cleavable linker P2A and the truncated hCD19as reporter.

CARs constructions were grafted in different effector host cells: Jurkatcell line (human CD4+ T cells) and NKT1.2 (murine iNKT) and CAR surfaceexpression was evaluated by flow cytometry and immunofluorescencemicroscopy. 87.6% of Jurkat HLA-G-15E7 and 83.2% of HLA-G-LFTT-1 CARtransduced cells express the chimeric receptors on their surface,detected by an anti-Flag antibody demonstrating that effector cellsstrongly expressed CARs constructs (FIG. 5A). Similarly, NKT 1.2HLA-G-15E7 and HLA-G-LFTT-1 CAR strongly expressed HLA-G CARs at theirsurface since 85.2% and 85.1% were respectively stained with theanti-Flag antibody. CARs cell surface expression was also determined byimmunofluorescence microscopy as shown in FIG. 5B.

Activation Assay: Membrane Acquisition through CAR/HLA-G1 Interaction

It was previously demonstrated that immune cells acquire cell surfacemembrane patches from target cells such as APC or tumor cellsantigen-presenting cells. Membrane acquisition from target cells byeffector cells (i) require cell-to-cell contact, (ii) is rapid, and(iii) is an active process dependent on the activation state of theacquirer cells which (iv) reflects the antigen-specific activation ofthe effector cells. Therefore, membrane transfers are related to theactivation of functionally mature immune cells.

Based on these principles, a test was designed to determine theactivation degree of the effector cells when they were confronted to thetarget subject: HLA-G molecules. The experiment was set up by using theJurkat T cell line as effector and target cells allowing us to avoid anybystander protein interaction. This implies that cell-to-cell contactwould only be mediated by the CAR-antigen interaction. Only those cellstransfected with the anti-HLA-G CAR are expected to be activated, thus,will present membrane acquisition when challenged with the target cells,and very low or no membrane transfer for control cells (Jurkat wt), asrepresented in FIG. 6A.

To characterize the isoforms predominantly expressed on HLA-G transducedJurkat target cells, HLA-G isoforms expression was determined by flowcytometry using the anti-HLA-Gβ2M-associated MEM-G/09 antibody and theanti HLA-G-α3 β2M-free 15E7 antibody. FIG. 6B show that Jurkat HLA-G1target cells only express HLA-G1 β2M-associated isoforms, and nostaining is observed following 15E7 staining, implying that no HLA-G-α3β2M-free isoform is expressed.

Then the level of membrane transfer was assessed, otherwise said, thedegree of activation of effector cells challenged with their targets.Membrane acquisition was 5.91% and 5.38% for Jurkat HLA-G-15E7 andHLA-G-LFTT-1 CAR cells when they were confronted to control Jurkat wt.However, given the nature of the HLA-G isoforms expressed on the targetcells (HLA-G1 B2M-associated), only HLA-G-LFTT-1 CAR are expected tospecifically bind and ergo activate the effector cells. Indeed JurkatHLA-G-LFTT-1 CAR acquisition increased to 21.7% whereas JurkatHLA-G-15E7 CAR activation remained at 5.6%. These results are shown inFIG. 6C.

Afterwards, the activation degree of HLA-G-15E7 CAR and HLA-G-LFTT-1 CARcells challenged with specific target cells was studied, based on the“Activation Index” ratio setting the baseline on the controlnon-specific cells: Jurkat wt cell line. As shown in FIG. 6D, JurkatHLA-G-LFTT-1 CAR is activated more than 3 times when challenged withJurkat HLA-G1Lv. However, activation does not increase when they arechallenged with Jurkat controls, neither HLA-G-15E7 CAR cells areactivated when challenged with non-specific targets (Jurkat wt andJurkat HLA-G1Lv).

Finally, the activation status of other immune cell subsets was assessedin order to confirm the specificity and biological activity ofHLA-G-15E7 CAR and HLA-G-LFTT-1 CAR constructions. As shown in FIG. 7A,Jurkat HLA-G-15E7 CAR was challenged with a specific target: K562HLA-G1p (that express mostly β2M-free isoforms of HLA-G) using K562 wtcells as control. Again, an increase of more than 3 times of activationwas observed. Also, NKT 1.2 HLA-G-15E7 and HLA-G-LFTT-1 CAR cells wheretested, challenged with Jeg-3 (human choriocarcinoma cells, expressingHLA-G1 β2M-associated isoforms). Only HLA-G-LFTT-1 CAR cells weresignificantly activated compared to NKT 1.2 HLA-G-15E7 or NKT 1.2control cells, as shown in FIG. 7B.

Cytolytic Function and IFN-γ Secretion of HLA-G CAR T Cells

Cytotoxic function of 15E7 and LFTT-1 CAR expressing effector cells wereassessed against JEG-3, K562 and K562-HLA-G1 transfected cell lines(FIGS. 8 et 9). To do so, target cells were labelled with CFSE beforebeing co-cultured 24 h with CAR-T cells at the indicated Effector:Targetratios (E:T). 24 h after coincubation, the medium was collected todetermine the IFN-γ secretion and cells recovered to investigate targetcell lysis by flow-cytometry.

Independently of the hinge used, K562 cell line was not lysed by antiHLA-G LFTT-1 and 15E7 CAR-T cells since K562 cells did not express HLA-Gprotein at their surface (FIG. 8A). K562-HLA-G1P cell line expressesboth HLA-G1 32M associated and β2M free isoforms at their surface (FIGS.2B and 2C respectively) and were almost completely lysed by LFTT-1 and15E7 CAR-T cells at E:T ratio of 10:1 in comparison to activated but nottransduced T cells. IgG4+CH3 hinge 15E7 CAR-T and LFTT-1 cells displayeda slightly lower efficiency in comparison to their IgG4 or IgG4+mCH2-CH3counterparts. CD107a expression was determined on effector CAR-T cellsaccordingly to their cytotoxic function (FIG. 8B). JEG-3 cell line onlyexpressed HLA-G1 associated to β2M isoform (FIG. 2C) and LFTT-1 CAR-Tcells were capable to lyse JEG-3 tumor cells (FIG. 8A). CD107aexpression was only determined on LFTT-1 CAR-T cells (FIG. 8B).

As shown on FIG. 9, against the HLA-G negative K562 cell line, nosecretion of IFN-γ was determined for LFTT-1 and 15E7 CAR-T cellswhereas both CAR-T cells strongly secrete IFN-γ following incubationwith the K562-HLA-G1P cell line. Against the JEG-3 cell line, onlyLFTT-1 CAR-T cells secreted IFN-γ, consistent with JEG-3 cells lysis andCD107a cell-surface expression on LFTT-1 CAR-T cells (FIG. 8).

In Vivo Experiment in NGS Mice

To investigate HLA-G CAR-T cells functions in vivo, K562-HLA-G1P tumorcells expressing the luciferase (K562-HLA-G1P-luc) were implanted in NGSmice followed by HLA-G CAR-T cells inoculation to monitor HLA-G CAR-Tcells cytotoxicity against HLA-G tumor cells (FIG. 10).

To do so, activated control cells and HLA-G CAR transduced T cells weregenerated as previously described. Briefly, human T cells were sorted,activated then transduced or not with either 15E7-IgG4 hinge-CAR orLFFT1-IgG4 hinge-CAR constructs and maintained in culture until day 9post-transduction to return to a resting state prior to in vivoexperiments.

Then, 18 NGS female mice were irradiated 24 h before K562-HLA-G1P-luccells intravenous inoculation. 3 days after K562-HLA-G1 cellsinoculation, 3 groups of 6 mice were established corresponding to: (i) 6mice were inoculated with 1.106 activated but not-transduced human CD8 Tcells (control group), (ii) 6 mice were inoculated with 1.10615E7-IgG4-CAR-T cells and (iii) 6 mice were inoculated with 1.106LFTT1-IgG4-CAR-T cells. K562-HLA-G1P-luc tumor growth was then monitoredto evaluate HLA-G CARs T-cell cytotoxicity against T cells control.

Material and Methods

Cells Lines

Jurkat cell line is human CD4+ T cells purchased from the ATCC (AmericanType Culture Collection TIB-152). Jurkat cell line was transduced withHLA-G1, HLA-G-LFTT-1 or HLA-G-15E7 CAR lentivirus, respectively. Jurkatwt and Jurkat transduced cell lines were cultured in RPMI 1640(Invitrogen) supplemented with 2 mM L-glutamine, 1 mg/ml penicillin andstreptomycin (Gibco), and 10% heat-inactivated FCS (Invitrogen).

NKT1.2 is a hybridoma murine cell line that was kindly provided by Dr.Kronenberg. These were also transduced with either HLA-G-LFTT-1 orHLA-G-15E7 CAR lentivirus respectively and were cultured in X-Vivo 10(Lonza) supplemented with 1 mg/ml penicillin and streptomycin (Gibco).

K562 cells are human leukemia cells purchased from the ATCC (AmericanType Culture Collection CCL-243). K562-HLA-G1p cells were obtained bynucleofection of K562 wt cells with an HLA-G1 encoding vector andK562-HLA-G1Lv cells were obtained by HLA-G1 encoding lentivirustransduction. These cell lines were cultured in RPMI 1640 (Invitrogen)supplemented with 2 mM L-glutamine, 1 mg/ml penicillin and streptomycin(Gibco), and 10% heat-inactivated FCS (Invitrogen).

Jeg-3 cell line is human choriocarcinoma cells purchased from the ATCC(American Type Culture Collection HTB-36). These were cultured in MEM(X) supplemented with 1 mg/ml penicillin and streptomycin (X), and 10%heat-inactivated FCS (Invitrogen).

HLA-G CAR Jurkat Cells

HLA-G-LFTT-1 and HLA-G-15E7 CAR constructs were generated as previouslydescribed [Pule et al. 2005 Molecular Therapy]. Briefly, theanti-HLA-G-recognizing domain is a single-chain variable fragment (scFv)derived from HLA-G1/β2M associated specific antibody LFTT-1 or from theHLA-G1/β2M free specific antibody 15E7. A short spacer derived from theIgG1 hinge region was used to link this scFv to the transmembranedomain. The HLA-G CAR endodomain was constituted by the fusion of CD28,OX40 and CD3ζ activation molecules. CAR construct was cloned into apTrip plasmid vector by digestion/ligation after extraction by PCR withspecific primers, under CMV immediate early promoter. Amino acidicsequences coding for the light and heavy chains of the scFv of Mabs aredetailed in SEQ ID NOs: 1 (VH), 2 (VL) and 31 (scFV) for LFTT-1 and inSEQ ID NOs: 3 (VH), 4 (VL) and 30 (scFV) for 15E7. The CAR sequences aredisclosed in SEQ ID NOs: 83 and 84 for the 15E7 CAR (amino acid sequenceand nucleic acid encoding sequence, respectively) and in SEQ ID NOs: 85and 86 for the LFTT-1 CAR amino acid sequence and nucleic acid encodingsequence, respectively).

HLA-G Jurkat Cells

HLA-G-expressing stable Jurkat cell line was generated by transductionand the lentiviral particles were generated as follows: specificsequences corresponding to native HLA-G1 cDNA (NM_002127.5) modifiedK334A and K335A according to Zhao et al. were cloned separately into apTrip plasmid vector by digestion/ligation after extraction by PCR withspecific primers, under CMV immediate early promoter.

Lentiviral Vectors

HIV-1-derived vector particles were produced by calcium phosphateco-transfection of HEK-293T cells (ATCC) with the recombinant plasmidpTRIP, an envelope expression plasmid encoding the glycoprotein fromVSV, serotype Indiana glycoprotein, and the p8.74 encapsidation plasmid.Viral stocks were titrated by real-time PCR on cell lysates fromtransduced HEK-293T cells and expressed as transduction unit (TU) perml. To generate Jurkat HLA-G-LFTT-1, HLA-G-15E7 CAR cells and JurkatHLA-G, 1×10⁵ Jurkat cells were seeded in 12-well plate with in 500 μl ofcRPMI medium and 10⁶ TU (293T) of Trip CMV-HLA-G-LFTT-1-CAR, TripCMV-HLA-G-15E7-CAR or Trip CMV-HLA-G vectors respectively. Cells wereincubated for 1 hour at 37° C. and then centrifuged 1 hour at 37° C.1200 g. Afterwards, 1 ml of cRPMI medium was added and incubated at 37°C. Two weeks later, positive cells were sorted by flow cytometry usinganti-HLA-G antibodies. The expression of HLA-G was evaluated by flowcytometry before the Jurkat HLA-G-CAR activation assay. The sameprocedure was carried out to obtain K562 HLA-G1Lv and NKT1.2HLA-G-LFTT-1 CAR or HLA-G-15E7 CAR cells.

Flow Cytometry

PE-conjugated mouse IgG1 (Clone P.3.6.8.2.1. 12-4714) from eBiosciences(Paris; France), FITC-conjugated rat IgG2a from BD Biosciences (clone:R35-95 553929, Le Pont de Claix; France) and MEM-G/09 (Exbio, Praha).Flow cytometry was carried out incubating the corresponding cell linewith monoclonal antibodies for 1 h at RT, washing twice and incubatingfor 30 min at RT with PE-conjugated goat anti-mouse IgG antibody(405307, Biolegend, USA). Flow cytometry analyses were performed usingLSR FORTESSA (Beckton Dickinson, Le Pont-de-Claix, France); data wereanalyzed with FlowJo X software (Tree star, Ashland, USA). The % ofpositive labeled populations was defined as those with stainingintensity higher than those exhibited by 99% of the isotype control.

Activation Assay

Jurkat HLA-G CAR effector cells and either Jurkat or Jurkat HLA-G1 tumorcells were respectively labeled with PKH26 and PKH67 fluorescent dyes(Sigma) according to the manufacturer's specifications. For activationassays based on the trogocytosis principle, Jurkat HLA-G-LFTT-1 orHLA-G-15E7 CAR (“acquirer” cells) were co-cultured with Jurkat or JurkatHLA-G1 cells (“donor” cells) for 1 h at a 1:1 effector-tumor ratio, in atotal concentration of 2×10⁶ cells/mL, and at 37° C. in a 5% CO₂humidified incubator as previously described [Caumartin J et al, (2007)Trogocytosis-based generation of suppressive NK cells, EMBO J26:1423-33]. At the end of the coincubation, cells were placed on iceand all further steps were performed at less than 4° C. Acquisition oftumor cell membrane by CAR effector cells was investigated by flowcytometry.

Vector Production

HIV-1 derived vector particles were produced by transient calciumphosphate co-transfection of HEK 293 T cells (ATCC) with the vectorplasmid pTRIP (encoding the glycoprotein from VSV, serotype Indiana(IND)) and the p8.74 encapsidation plasmid for the production ofIntegrative Lentiviral Vector particles (ILV). Vector gene transfercapacity was determined by quantitative PCR after transduction of 293Tcells as previously described (Coutant, F., et al., PLoS One, 2008.3(12): p. e3973) and was expressed as transduction unit (TU)/mL ofvector.

T Cell Isolation and Activation

PBMCs were extracted from blood sample of 3 different healthy donors(EFS, Rungis) after ficoll isolation. T cells were sorted by columnpurification (Miltenyi), activated with CD3+ CD28+ coated microbeads(Miltenyi) and then cultured 48 hours at 37° C., 5% CO₂ in RPMI 1640Glutamax (Gibco) supplemented with 10% FCS 1% Penicillin-Streptomycin(Gibco), 50 uM Beta-mercaptoethanol (Gibco), Non Essential amino acid,10 mM Hepes (Gibco) and 1 mM Sodium Pyruvate (Gibco). Then, cells werewashed and transduced with lentiviral vectors at MOI 20 in 200 μl at 1M/ml during 4 hours under slow shaking. Cells were then transferred in aU-bottom 96-wells plate. After 24 h cells were adjusted at 1.10⁶cells/ml and 50 U/ml of human IL2 were added (Preprotech). Every 2-3days, cells were counted and adjusted at 1.10⁶ cells/ml in completemedium with IL2. After 8 days, cells were used for function assays.

Cytotoxicity Assays and Activation Profile

Cytotoxic assays were performed against JEG-3, K562 and K562-HLA-G1target cells.

Briefly, 24 hour prior to the assay, 3.10⁵ JEG-3 cells were labelledwith CFSE (CellTrace, Thermofisher) at a 1/10 000; 3.10⁵ K562 or 3.10⁵K562-HLA-G1 cells were labelled with CFSE at a 1/20 000 dilution. Targetcells were then plated in a U-bottom 96-wells plate. CAR T cells werewashed in PBS 1× (Gibco) before being co-cultured with target cells atthe indicated Effector:Target ratios (E:T).

After 24 h of coincubation, medium was collected and cells recovered(detached in the case of JEG-3) in PBS-EDTA 0.1%. After washing, cellswere labelled with antibodies against: CD4 (clone SK3 Percp, BDPharmingen), CD8 (clone SK1 PE-Cy7, Biolegend), CD19 (clone LT19 FITC orPE, Miltenyi), CD25 (clone M-A251 BV421, BD Horizon), CD69 (clone FN50BV711, BD Horizon), PD-1 (clone EH12.2H7 APC, Biolegend) and Live/dead(eFluor 780, Thermofisher) following manufacturer recommendations.Acquisition was performed with an Attune cytometer (Thermofisher) andresults were analyzed with FlowJo software.

IFNγ Secretion Assays

IFNγ quantification was performed directly on co-culture medium using aCBA kit (BD Biosciences) following the manufacturer instructions.

Degranulation Assay

Co-cultured cells were prepared as described previously (E:T ratio of10) and the anti-CD107a (clone H4A3 PE, Biolegend) was directly added tothe co-culture. 1 hour after the co-incubation, Monensin (GolgiStop, BDBioscience) was added. Cells were recovered 5 hours after co-incubationexperiment and labelled with CD4, CD8, CD19 and Live/dead antibodies.Acquisition was performed with an Attune cytometer and results wereanalyzed with FlowJo software.

In Vivo Experiment

NOD/SCID/IL2Rγc-deficient (NSG) mice (6-8 weeks of age, The Jacksonlaboratory, Sacramento, Calif., USA) were irradiated (2.5 Gy) andinoculated intravenously with appropriated number of luciferaseexpressing K562-HLA-G1P (1.106/mouse) tumor cells. T cells (untransducedand transduced) were injected in the tail vein on day 3 forK562-HLA-G1P-Luc model. Engraftment was monitored every 3 to 4 daysuntil day 17 post-implantation and then weekly by BLI measurements: micereceived intraperitoneal 3 mg of luciferin (VivoGlo Luciferin, #P1043,Promega, Wisconsin, USA) within 10 minutes before imaging (IVIS LuminaSeries III, Perkin Elmer, Massachusetts, USA).

Statistical Analyses

Data are presented as means +/−standard deviation (SD). Student t testwas used and a P value less than 0.05 was taken to be significant. Forfigures showing representative experiments, error bars represent SD oftriplicates.

CONCLUSION

Considering that the basic criteria to develop CARs immunotherapy arethe identification of proper Tumor Associated Antigen, the accessibilityof transgenic effector cell and reversibility of the immunosuppressivetumor microenvironment, it seems clear that HLA-G represents a veryinteresting candidate for this purpose. HLA-G is considered an ICPbecause of its key role on immune modulation, it has also beenextensively described that this molecule is expressed in numerous tumoreffusions of diverse origins, and no cellular or humoral response hasever been described against it. In addition, HLA-G is a singularmolecule for which its function has been characterized but its proteinstructure turns out to be very complex and heterogeneous. Thus, solelyone approach wouldn't be enough to inhibit HLA-G function.

Here is proposed the generation of two CARs directed against HLA-G usingthe scFv of anti-HLA-G monoclonal antibodies (Mabs), directed todifferent epitopes of the molecule aiming most of HLA-G isoforms:classical HLA isoforms and smaller β2M-free isoforms. Therefore, it isexpected to eliminate most of the cells that express HLA-Gimmunosuppressive isoforms. Effector cells transduced with the nucleicacid construct according to the invention were generated and these CARmolecules are highly expressed at the cell surface and are biologicallyfunctional. Also, anti-HLA-G CAR NKT cells were generated. It waspreviously reported that NKT cells were better immune cells for CARtherapy than T or NK counterparts. Furthermore, it was shown that CARNKT cells infiltrate with a higher efficiency solid tumor. Since solidtumor cells highly express HLA-G (Paul et al. 1999 Cancer Research,Rouas-Freiss et al. 2007 Semin Cancer Biol, Yaghi et al. 2016Oncotarget), anti-HLA-G NKT CAR might be an innovative way to circumventthe immunosuppressive environment linked to HLA-G.

To summarize, here is demonstrated that the 3^(rd) generationrecombinant construction CARs according to the invention (i) arecorrectly expressed on the surface of the transduced cells, (ii) thateach CAR: HLA-G-15E7 CAR and HLA-G-LFTT-1 CAR are specific for HLA-Gβ2M-free or β2M-associated immunosuppressive isoforms respectively and(iii) that the CAR expressing effector cell against HLA-G are properlyactivated.

The invention claimed is:
 1. A cell comprising a chimeric antigenreceptor (CAR) comprising: (a) an extracellular domain comprising anantigen binding domain that specifically binds to HLA-G, wherein theantigen binding domain comprises: (i) a heavy chain variable region (VH)comprising a heavy chain complementarity determining region 1 (HC CDR1)of SEQ ID NO: 5, a heavy chain complementarity determining region 2 (HCCDR2) of SEQ ID NO: 6, and a heavy chain complementarity determiningregion 3 (HC CDR3) of SEQ ID NO: 7; and (ii) a light chain variableregion (VL) comprising a light chain complementarity determining region1 (LC CDR1) of SEQ ID NO: 8, a light chain complementarity determiningregion 2 (LC CDR2) of SEQ ID NO: 9, and a light chain complementaritydetermining region 3 (LC CDR3) of SEQ ID NO: 10; (b) a CD28transmembrane domain; and (c) an intracellular domain comprising a 4-1BBcostimulatory signaling region and a CD3 zeta endodomain.
 2. The cell ofclaim 1, wherein the cell is a T cell, a B cell, a NK cell, a NKT cell,a monocyte cell or a dendritic cell.
 3. The cell of claim 1, wherein theVH comprises SEQ ID NO: 1 and the VL comprises SEQ ID NO:
 2. 4. The cellof claim 1, wherein the antigen binding domain is a scFv.
 5. The cell ofclaim 1, wherein the antigen binding domain comprises SEQ ID NO:
 31. 6.The cell of claim 1, wherein the CAR further comprises a human IgG4hinge domain connecting the antigen binding domain to the transmembranedomain.
 7. The cell of claim 1, wherein the CAR comprises SEQ ID NO: 32,SEQ ID NO: 33, or SEQ ID NO:
 34. 8. The cell of claim 1, wherein theCD28 transmembrane domain comprises SEQ ID NO: 20, wherein the 4-1BBcostimulatory signaling region comprises SEQ ID NO: 21, and wherein theCD3 zeta endodomain comprises SEQ ID NO:
 22. 9. The cell of claim 1,wherein the intracellular domain further comprises a CD28 costimulatorydomain.